ALBERT

Description: In MM patients relapsing after MRD-negativity, the disease could reemerge from immature cells or from undetectable MRD. However, it remains unknown if immature cells have the same genetic background as MM plasma cells (PCs), as well as the amount of MRD that persists below the limit of detection (LOD) of next-generation techniques. To obtain further insight, we compared the biological landscape of MM PCs at diagnosis to that of CD34 progenitors, B cells and normal PCs isolated from patients with negative MRD by next-generation flow (NGF) after treatment. We performed whole-exome sequencing (WES, mean depth: 90x) with the 10XGenomics Exome Solution for low DNA-input as well as deep NGS of B-cell receptor immunoglobulin (BcR IG) gene rearrangements (mean, 69,975 sequences), in a total of 68 cell-samples isolated from the bone marrow (BM) of 7 MM patients with MRD-negativity by EuroFlow NGF after induction with VRD and auto-transplant (GEM2012MENOS65 trial). Patients with negative MRD were intentionally selected to avoid contamination with MM PCs during sorting of CD34 progenitors, B-cell precursors, mature B cells and normal PCs after induction and transplant. We investigated in these populations the presence of somatic mutations and clonotypic BcR Ig rearrangements detectable in MM PCs sorted at diagnosis, using peripheral blood T cells as germline control. We also performed WES in matched diagnostic MM PCs and MRD cells persisting after VRD induction in 14 cases as control. In another 6 patients with untreated MM, we performed single-cell RNA and BcR IG sequencing (scRNA/BcRIGseq) of total BM B cells and PCs (n=16,380) to investigate before treatment, if the clonotypic BcR IG sequence of MM PCs was detectable in other B cell stages defined by their molecular phenotype. We used multidimensional flow cytometry (MFC) to investigate the frequency of B cell clonality in BM samples from a larger series of 195 newly-diagnosed MM patients, prospectively enrolled in the GEM-CLARIDEX trial. Somatic mutations present in diagnostic MM PCs were detectable in the lymphopoiesis of 5/7 patients achieving MRD-negativity after treatment. In one case, out of 55 mutations present in diagnostic MM PCs, a single mutation in PCSK1N (VAF: 0.30) was detectable in normal PCs. In the other four patients, a total of 85 mutations were present in MM PCs and up to 10 (median VAF, 0.16) were found all the way from CD34 progenitors into B-cell precursors, mature B cells and normal PCs, but not in T cells. Of note, most mutations were reproducibly detected in each cell type after induction and after transplant. All somatic mutations shared by MM PCs and normal cells were non-recurrent, and genes recurrently mutated in MM (eg. ACTG1, ATM, DIS3, FAM46C, KRAS, LTB, MAX, TRAF3) were found in MM PCs but never in normal cells. Copy number alterations (CNA) were found only in MM PCs. By contrast, up to 513/827 (62%) mutations and 48/67 (72%) CNA were detectable in matched diagnostic MM PCs and persistent MRD cells, indicating that the few somatic variants present in normal cells were unlikely related to contaminating MRD below NGF's LOD. Accordingly, MM clonotypic BcR IG rearrangements were detectable in normal PCs (4/7patients) and in immature B cells (5/7 patients) but at much lower frequencies (mean of 0.02% in both). Of note, 9 additional clonotypes (mean 8.4%) were found in MM PCs of 5/7 patients (range, 1-3). scRNR/BcRIGseq unveiled that clonotypic cells were confined mostly but not entirely within PC clusters, and that in 1 patient another clonotype was detectable in mature B cells. Accordingly, using MFC we found in a larger series that 25/195 (13%) of newly-diagnosed MM patients display B-cell clonality (median of 0.7% BM clonal B cells, range 0.02%-6.3%). In conclusion, we show for the first time that MM patients bear somatic mutations in CD34 progenitors that specifically differentiate into the B cell lineage, likely before the disease onset. Because diagnostic, MRD (and relapse) MM PCs display great genetic similarity, these results suggest that undetectable MRD

Description: Investigation of minimal residual disease (MRD) in acute leukemias by immunophenotyping is increasingly used for disease monitoring. Most MRD studies using flow cytometry techniques have focused on patients treated with conventional chemotherapy, while information on its value in patients undergoing allogeneic transplantation is scanty. The aim of the present study is to evaluate whether or not immunophenotypical assessment of MRD could also be a valuable tool in patients undergoing allogeneic transplantation for acute leukaemia. For that purpose we have analysed the level of MRD before and after (month +3) transplantation, by multiparameter flow cytometry, in a series of 38 acute leukaemia patients (26 ALL cases -20 cases in 1st CR and 6 in 2nd CR- and 12 AML cases -all of them in 1st CR), that showed an aberrant immunophenotype at diagnosis. Although the level of MRD in the BM obtained before transplantation showed a tendency to predict RFS, differences did not reached statistical significance. By contrast, the evaluation of the BM obtained 3 months after allo-transplantation had significant prognostic value. Thus, patients with low MRD levels (

Description: Chronic myelomonocytic Leukemia (CMML) is a heterogeneous clonal disorder highly resistant to the few therapies that are available nowadays. There is increasing evidence to suggest that Programmed Death-1 (PD-1), and its major ligand Programmed Death Ligand-1 (PD-L1), are involved in immune suppression and disease progression, are highly expressed in many hematological malignancies, and can be involved in MDS pathogenesis and resistance mechanisms to hypomethylating agents. However, the expression of PD-1 and PD-L1 is not widely explored in CMML. Different types of monocytes based on CD14 and CD16 expression show different genetic profiles and functions, having different distribution in several conditions, including malignancy. In our study, we studied the expression of PD-1 and PD-L1 in the peripheral blood (PB) monocytic compartment of patients with CMML using flow cytometry , to better understand their potential role in the pathogenesis of the disease, and as a basis for the evaluation of this pathway for the development of future immunotherapy strategies. Peripheral blood samples from 16 CMML, and age matched normal (n=10) and reactive (n=9) monocytosis (〉1 x109 /L) were studied. Two hundred µl of each PB sample were stained with an 8-color panel of monoclonal antibodies (CD16-FITC, CD64-PE, PD1-PCP5.5, PDL1-PC7, CD300-APC, CD14-APCH7, HLADR-V450 and CD45-OC515). A minimum of 1x 106 events were acquired by FACSCanto II (BD Biosciences, San Jose, USA) and the data was analyzed with the Infinicyt software (Cytognos SL, Spain). Monocytic population was selected first on the automated population separator plot and confirmed by the expression of CD64 and HLADR expression. Lymphocytes were used as the internal control. Three types of monocytes were defined based on the CD14 and CD16 expression, as previously described. As expected, CMML type 1 patients had higher absolute monocyte counts in PB than reactive and normal cases (p=0.001), and higher percentage of monocytic cells by flow (0.001). The distribution (median) of the monocytic subpopulations based on CD14 and CD16 expression among the monocytic compartment in PB of CMML, reactive and normal cases, respectively, was as follows: "classical"(CD14+CD16-) were of 98%, 90% and 85% (p

Description: Abstract 4051 Multiparameter flow cytometry (MFC) immunophenotyping has shown to be of value for differential diagnosis and minimal residual disease assessment in multiple myeloma. However, the clinical value of MFC immunophenotyping in other plasma cell disorders (PCD) remains largely unexplored. Systemic light chain (AL) amyloidosis is a rare PCD characterized by the accumulation of monoclonal light chain fragments leading to end-organ damage and short survival. Bone marrow (BM) plasma cell (PC) infiltration in AL is usually low and thus the identification of clonal PC can be often difficult by immunohistochemistry and/or immunofluorescence. In the present study we focused on 34 BM samples sent to our institution with a suspected diagnosis of AL. MFC immunophenotypic studies were performed using the following 4-color combinations of MoAbs (FITC/PE/PerCP-Cy5.5/APC): CD38/CD56/CD19/CD45 (n=34); in addition cy-Kappa/cy-Lambda/CD19/CD38 staining was add to confirm the clonal or polyclonal nature of BMPC in equivocal cases. Ploidy and cell cycle analysis were additionally performed in a subset of cases (n=12/34). From the total 34 cases included in the present study, 28 had a confirmed diagnosis of AL. The remaining 6 cases were finally diagnosed with localized - amyloidoma - (n=2) and familial (n=1) forms of amyloidosis, multiple myeloma-associated amyloid (n=2) and congestive pericarditis (n=1). Interestingly, the presence of clonal PC was detected by MFC in 27 of the 28 (96%) patients with AL; in turn, clonal PC were undetectable in the BM of all cases with localized and familial forms of amyloidosis. The median overall level of PC (M-PC plus N-PC) seen in MFC immunophenotypic analyses of BM samples of the 28 patients with AL was 1.9% (range: 0.1% - 15%), with a significant positive correlation between PC enumerated by MFC and conventional morphology (r=0.5; p=.01). Within the BMPC compartment, the median proportion of clonal PC was of 94% (mean 81% ± 29%); in 6 cases all BMPC were clonal while in the remaining 22 patients residual normal PC persisted (median of normal PC/BMPC 13% ± 31%). The most common aberrant phenotypes were down-regulation of CD19 (92%) and CD45 (83%), followed by overexpression of CD56 (56%) and infra-expression of CD38 (42%). Aneuploidy was only found in 18% of cases, all of them hyperdiploid. Cell cycle analysis showed a median % of S-phase and G2-Mitosis PC of 0.7% and 3.5%, respectively. Concerning patients' outcome, cases with undetectable normal PC (6/28, 21%) had a significantly decreased overall survival (OS) compared to patients with persistent BM normal PC at diagnosis (22/28, 79%) with 3-year OS rates of 0% vs. 59%, respectively (p=.001). In summary, these preliminary data suggests that MFC immunophenotyping investigations may be clinically relevant in patients with suspected amyloidosis for i) differential diagnosis between AL and other forms of amyloidosis and, ii) prognostication of patients with AL according to the presence or absence of baseline persistent normal PC. Disclosures: No relevant conflicts of interest to declare.

Description: Recently, a new set of parameters produced by the Beckman-Coulter’s GEN’S, LH500, LH750 and the latest LH780 automated blood counters, derived from leukocyte and red cell’s morphological data by means of the VCS technology are available and can be printed as research data together with the leukocyte differential and reticulocyte analysis. With the use of these Research Population Data (RPD) parameters several recent publications have shown their usefulness for the diagnosis of malaria (Fourcade et al), sepsis (Chaves ), apoptosis detection (Lab hem 2005) and the diagnosis and differentiation of lymphoptoliferative disorders (Silva M et al) In order to evaluate the analytical behaviour of these newly described red blood cell and leukocyte morphological parameters in routine bood samples and in the Beckman-Coulter’s calibration and control 5C, we conducted a study to evaluate the paramters’ precision and performance during short and long term sample’s storage: Precision: 10 samples from normal individuals were analyzed 10 times and the CV for each parameter was calculated Short term stability stability at 4C and 22C: 5 normal samples were evaluated from the time of blood drawing until 5 hours later, both at room temperature (22C) and at 4 C Long term stability at 4C: 15 normal samples were evaluated at various times after blood drawing, from 4 hours (which was found to be the best time for sample analysis in the short term stability study, above) up to 77 hours after blood withdrawal. Control and Calibration with 5C: We evaluated the performance of the research parameters in the 5C by running the same batch of 5C control in 5 different counters from 4 different centers, comparing the results with those from 5 normal individuals Stability in a period of time: We have evaluated every week during two months 15 normal samples, recording the temperature and the avergae of all RPD values. RESULTS Precission: The CV% for the Mean Volume, Conductivity and Scatter of the different leukocyte types was always less than 1%, with an average of 0,6%. The CV% for the Standard Deviation of Volume, Conductivity and Scatter varied between 2.45% and 7.7%, wit an average of 4.9%. The CV% of any SD always being higher than the primary parameter. Short term stability: We observed no significant differences between samples kept at 4C and 22C during the first 5 hours after blood drawing. During the first two hours, however, minor fluctuations in results occurred, always

Description: Proteasome inhibition represents a promising novel anticancer therapy, and Bortezomib is a highly selective reversible inhibitor of the 26S subunit of the proteasome complex. Acute Myeloid Leukemia (AML) is a heterogeneous group of diseases in which coexistence of leukemic cells at different stages of differentiation is frequently found, and CD34, a marker of immaturity, has been associated with drug resistance and poorer outcome. In order to explore the activity of Bortezomib in AML, we have evaluated its effectiveness against freshly isolated cells from 27 newly diagnosed AML patients (excluding APL). Mononuclear cells (MNC) were isolated from fresh bone marrow samples by Ficoll-Hipaque density sedimentation. The percentage of blasts after purification was 88±9% (Mean±SD). In order to assess the effective dose of Bortezomib in AML, four cell lines (HL-60, KG-1, HEL y MV4-11) were treated for 24h with increasing doses of Bortezomib. IC50 was 50nM (Range 10nM to 100nM). Based on these results, patients’ samples were treated with Bortezomib for 18 hours. Doxorubicin 1μM was also used for treatment in order to compare with Bortezomib activity in 23 paired samples. Using quadruple staining (Annexin V/CD33/CD34/CD45), the number of apoptotic cells was measured by multiparametric flow cytometry. At least 50.000 events were acquired in a FACScalibur cytometer (BDB). Annexin V+ (AV+) events were quantified over total cellularity, as well as in CD34+, CD34− blast cell fractions and normal residual lymphocytes. The percentage of apoptotic events was corrected according to the proportion of apoptotic cells in the control tube (absence of drug). The proportion of AV+ cells in the total blast cell population was 55±27%. CD34+ and CD34− blast cells populations were separately analyzed (only one leukemic cell population CD34+, or CD34− were detected in 7 and 13 cases, respectively; while in the remaining 6 cases both populations existed). Interestingly, Bortezomib showed similar activity on CD34+ (52±26%, n=13) and CD34− (55±26%, n=19) blast cell populations (p=0.68). Moreover, in the 6 cases in which CD34+ and CD34− leukemic cells coexisted, no difference was detected on the proportion of AV+ events after Bortezomib treatment in both cellular subsets (42±26% and 48±16% respectively, p=NS). In addition, the proportion of AV+ cells within the normal residual lymphocytes with bortezomid was significantly lower (20±12%) when compared with blast cells (p〈 0.01). Bortezomib was more effective for total blast cells compared to doxorubicin (56±32% vs 40±26%, respectively; p=0.009). Interestingly, when CD34+ and CD34− blast cell populations were separately analyzed, differences were only significant for the CD34+ populations (n=13) (52±26% vs 32±28% AV + cells for Bortezomib and doxorubicin, respectively; p=0.009), with no differences in either CD34− populations or normal residual lymphocytes. In summary, our results show a high antitumour activity of Bortezomib in AML blast cells, with similar efficacy against the more immature CD34+ and more mature CD34− leukemic cell subsets, while the toxicity against residual lymphocytes is significantly lower. These results support a therapeutic role for Bortezomib in adult Acute Myeloid Leukemia.

Description: The Internet is changing the way that people learn about health and illness. At present do not exist data of the use of Internet by patients of lymphoma and her caregivers in Spain. OBJECTIVE: To investigate the distribution and patterns of Internet use by patients with lymphoma and her caregivers. PATIENTS AND METHODS: 585 subjects (258 patients, 264 relatives and 63 health professionals), 228 male and 357 female, they have responded a questionnaire on diverse aspects of the use of Internet. RESULTS: Two hundred fifty (42,7%) subjects use Internet, although only 27% make to obtain data on lymphoma. With respect to the group of patients 31% recognize to use Internet, but only the 23,3% do it by questions related to their disease. The main reasons for Internet use are to obtain information about treatments (74.7%) or second opinion medical (9.3%). The 77,6% have been using Internet for more than 3 years; the 47,2% have university studies and the 58,4% have between 33–50 years. Mainly the information search is made in Spanish language and through the Google finder. They consider that Information on lymphoma is acceptable (44.9%) or of enough quality (43.7%), trustworthy (50.6%) or of enough reliability (33.5%) and useful (45.6%) or quite useful (37.3%). COMMENTARIES: This study contributes data on the use of Internet by patients with lymphoma and her caregivers in Spain. Oncologists should be familiar with this important resource to help patients access appropriate material.

Description: Acute myeloid leukemia (AML) is a malignant disease characterized by uncontrolled proliferation, differentiation arrest and accumulation of immature myeloid progenitors. Despite recent developments and the approval of new therapeutic agents in the last few years, long term survival of AML, particularly in elderly patients remains an unmet medical need.The use of all-trans retinoic acid (ATRA) in Acute Promyelocytic Leukemia has proven that differentiation therapy may significantly change the survival of AML patients, however the success in APL has not been translated to other groups of AML. Therefore, the identification of new therapeutic agents that may induce the differentiation of AML blasts represents an attractive new target. Furthermore, it is well known that epigenetic alterations have an important role in the development and maintenance of cancer and AML in particular. Thus, our aim was to develop new small molecules targeting epigenetic modifying enzymes like DNA methyltransferases (DNMT), histone methyltransferases or histone deacetylase (HDAC) with the aim of inducing differentiation in AML. We performed a screening of over 50 small molecules synthesized by our group. The design was performed in-house using a knowledge and structure based strategy and the read out of the screening was based on changes in expression of CD11b (a well described marker of myeloid differentiation) after in vitro treatment of AML cells lines. Interestingly, we found several compounds with high capacity to promote the differentiation of leukemic cells in AML cells lines at low non-cytotoxic doses, selecting CM-444 and CM-1758 as our lead compounds (Figure 1a).A complete biochemical characterization showed that both compounds are specific pan-HDACs inhibitors (HDACi). CM-444 and CM-1758 induced in vitro cell differentiation in all subtypes of AML, independently of the AML genetic subgroups or the presence of mutations, which was significantly more pronounced that differentiation induced by reference compounds such as Panobinostat, Vorinostat, Entinostat, Tubastatin or Quisinostat, previously described HDACi. CM-444 and CM-1758 also induced in vivo differentiation in xenogeneic models of AML. AML differentiation was associated with induction of CD11b, downregulation of c-MYC, overexpression of transcription factors that govern the myeloid differentiation and morphologic changes. In addition, these compounds promoted in vitro differentiation of patient-derived AML blasts. The complete transcriptome analysis by RNA-Seq carried out in AML cell lines after CM-444, CM-1758, Panobinostat or Vorinostat treatment showed changes in genes implicated in differentiation, but without explaining the differences among the different HDACi. Analysis of the complete acetylome and proteome before and after treatment with CM-444 and CM-1758 in comparison with other HDACi showed differential acetylation of non-histone proteins included in the GO categories of Zn binding proteins and nucleic acid binding proteins (Figure 1b). Most of these proteins are epigenetic enzymes and have been related to AML and myeloid differentiation, such as MLL2, EP300 or BRD4. In summary, we have developed and characterized novel epigenetic small molecules with a high in vitro and in vivo capacity of differentiating AML cells. These compounds might be an effective differentiation-based therapy to be tested in AML. Besides, the mechanism of differentiation of these compounds is due, at least in part, to the acetylation of non-histone epigenetic proteins, which are key in the myeloid differentiation. Disclosures Paiva: Celgene, Janssen, Sanofi and Takeda: Consultancy; Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria.

Description: Imatinib mesylate, a targeted inhibitor of BCR-ABL tyrosine kinase, is the standard of care for chronic myeloid leukemia (CML). A phase 2 trial of imatinib in late chronic-phase (CP) CML after interferon-α (IFNα) failure enrolled 532 patients, 454 with a confirmed diagnosis of CP CML. Median time from diagnosis was 34 months; median duration of imatinib treatment was 65 months. Cumulative best rates of major cytogenetic response (MCyR) and complete cytogenetic response (CCyR) were 67% and 57%, respectively. At the 5-year landmark, 184 (41%) of the 454 patients are in CCyR. At more than 6 years, 199 (44%) of the 454 patients remain on imatinib. Most responses occurred within 12 months of starting imatinib; however, some patients achieved initial MCyR and CCyR more than 5 years after imatinib initiation. Estimated rates of freedom from progression to accelerated phase (AP) and blastic phase (BP) and overall survival at 6 years were 61% and 76%, respectively. Both freedom from progression to AP/BP and overall survival (OS) were associated with cytogenetic response level at 12 months. No increase in rates of serious adverse events was observed with continuous use of imatinib for up to 6.5 years, compared with earlier time points. Imatinib continues to be an effective and safe therapy for patients with CP CML after failure of IFN.

Description: Minimal residual disease (MRD) assessment is standard in many hematologic malignancies but is considered investigational in multiple myeloma (MM). We report a prospective analysis of the prognostic importance of MRD detection by multiparameter flow cytometry (MFC) in 295 newly diagnosed MM patients uniformly treated in the GEM2000 protocol VBMCP/VBAD induction plus autologous stem cell transplantation [ASCT]). MRD status by MFC was determined at day 100 after ASCT. Progression-free survival (PFS; median 71 vs 37 months, P 〈 .001) and overall survival (OS; median not reached vs 89 months, P = .002) were longer in patients who were MRD negative versus MRD positive at day 100 after ASCT. Similar prognostic differentiation was seen in 147 patients who achieved immunofixation-negative complete response after ASCT. Moreover, MRD− immunofixation-negative (IFx−) patients and MRD− IFx+ patients had significantly longer PFS than MRD+ IFx− patients. Multivariate analysis identified MRD status by MFC at day 100 after ASCT as the most important independent prognostic factor for PFS (HR = 3.64, P = .002) and OS (HR = 2.02, P = .02). Our findings demonstrate the clinical importance of MRD evaluation by MFC, and illustrate the need for further refinement of MM re-sponse criteria. This trial is registered at http://clinicaltrials.gov under identifier NCT00560053.