ALBERT

Description: Background Myelodysplastic syndrome (MDS) is a pre-leukemia disease affecting the erythroid, myeloid and megakaryocytic bone marrow production. MDS patients are classified according to the WHO classification of myeloid neoplasms. During the past 15 years management of MDS patients has been stratified according to the International Prognostic Scoring System (IPSS) risk score. Recently a revised version of IPSS has been introduced (IPSS-R). One quarter of LR-MDS in this new IPSS-R were reclassified as having a higher risk and a substantial subset of high risk-MDS (HR-MDS) were reclassified as lower risk. In LR-MDS a differentiation block is observed in the erythroid lineage. The diagnosis and follow up of cytopenias in particular anemia must be the main objective (1). The soluble transferrin receptor (sTfR) directly reflects the erythropoietic activity in individuals without iron defiency and may appreciate ineffective dysplastic erythropoiesis in LR-MDS. In LR-MDS there is also an inverse relationship between EPO level and the degree of anemia but a wide range of EPO levels is found in patients with similar Hb concentrations. Thus the highest EPO levels are found in patients with erythroid hypoplasia in bone marrow. Aims The combination of several biomarkers: Hb, ferritin, EPO and sTfR may be useful in LR-MDS for diagnosis and follow up. Methods A total of 192 patients with LR-MDS were investigated. Median age of the 192 patients was 71 years (21-92) with 56% males, median survival: 54 months, median follow up: 102 months. The stratification according to the WHO criteria and IPSS risk score was realized. Bone marrows were studied and cytogenetic assessment was realized in the same time. Serum concentrations of ferritin, EPO and sTfR has been analyzed by immuno-assays. Hb level was determined on Beckman Coulter apparatus. The follow up of Hb, ferritin, EPO and sTfR was realized every 2 months in patients with supportive care only until the first specific treatment. A multivariate logistic regression analysis to ascertain the correlations between disease progression and studied biological parameters was realized. Results The logistic regression analysis of our results is significant to define a biological evolutive profile of LR-MDS patients with these biomarkers. The combination of these routine parameters may represent a functional erythropoietic follow up in LR-MDS patients (table 1). This biological tool is an easy method to observe the red cell lineage of LR-MDS patients. This combination informs about the progressive ineffective and dysplatic erythropoiesis in LR-MDS patients. The measurement of ferritin which is a correlated parameter in LR-MDS shows the level of iron overload. A normal or high level without inflammation condition excludes an iron deficiency. The EPO level can give a predictive information about the future efficacy of ESA (endogenous EPO 〈 500 U/l). Conclusion With our results and a correlative logistic regression analysis, we can propose a biological scoring system to appreciate the evolutive anemia of LR-MDS progression in patients. In LR-MDS the management of patients may be based on personalized medicine according a risk assessment with IPSS-R, cytogenetics, mutations and HLA typing (2). But an additional biological and functional predictive scoring system informs about the important independent role of dysplasias particularly anemia in LR-MDS patients before to choose a suitable therapy: transfusions, iron chelation, ESA, TGF-ï¢ pathway inhibitors, G-CSF, immun suppressive treatment, lenalidomide, azacytidine, allogeneic HSCT Table 1. Hb ± EPO ±  sTfRDysplastic erythropoiesis without anemia Hb ±  EPO  sTfRStabilized dysplastic erythropoiesis Hb  EPO  sTfRUnstabilized dysplactic erythropoiesis Hb  EPO  sTfRIneffective dysplastic erythropoiesis EPO 〈 500 U/l : ESA may be efficient〉 500 U/l : ESA will be inefficientFerritin level : iron overload References Giagounidis A Management of low-risk myelodysplastic syndromes Hematology Education, 2015, 9 (1), 219-225 Platzbecker U et al Personalized medicine in myelodysplastic syndromes: wishful thinking or already clinical reality? Hematologica, 2015, 100 (5), 568-571 Disclosures No relevant conflicts of interest to declare.

Description: An obstacle with continued clinical development of CAR T cells is the limited understanding of their biology and mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (Fig 1A) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ, IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytotoxicity was enhanced compared to non-mutated CARs (Fig 1B). Using a pre-clinical immunocompetent mouse tumor model, we found the mut06 CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells (Fig 1C). To examine exhaustion, we ex vivo stimulated CAR T cells with target cells expressing CD19 and PDL1 and found mut06 CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that mut06 CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. After 24hr stimulation with CD19 target cells mut06 CAR T cells had a significant reduction in pAkt compared to m1928z CAR T cells, which is a critical signaling mediator in the NFAT and NR4A1 transcription factor pathways. Additionally, mut06 had decreased p-NFAT compared to m1928z when examined by western blot. To determine how optimized CAR signaling affected T cell exhaustion we looked at 22 genes that are upregulated when NFAT is constitutively active and overlap with genes identified as important for T cell exhaustion. We found that most of the exhaustion related genes were upregulated in m1928z CAR T cells while they were decreased in m19hBBz. The mut06 CAR T cell gene expression pattern was more similar to m19hBBz with exhaustion related genes downregulated compared to m1928z (Fig 1D). To examine differences in the accessibility of exhaustion related genes we performed ATAC-seq and found NFAT (Nfatc1) and NR4A2 (Nr4a2) had lower chromatin accessibility profiles in mut06 compared to m1928z (Fig 1E). We also found that exhaustion related genes Havcr2 (TIM3), Pdcd1 (PD1), and Lag3 (LAG3) all had greatly reduced chromatin accessibility in mut06 CAR T cells compared m1928z. Overall, these genomic studies support our findings that mut06 optimizes CAR T cell signaling by lowering transcription factors that regulate exhaustion. Figure 1 Disclosures Li: ImmuneBro Therapeutics: Other: sole shareholder . Davila:Atara: Research Funding; Celgene: Research Funding; GlaxoSmithKline: Consultancy; Novartis: Research Funding; Anixa: Consultancy; Bellicum: Consultancy; Adaptive: Consultancy; Precision Biosciences: Consultancy.

Description: Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Description: Controversy exists as to whether Kaposi's sarcoma–associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Description: Patients with chronic lymphocytic leukemia (CLL) may develop other B cell malignancies in their clinical course including aggressive diffuse large B-cell lymphomas and rarely myelomas. In a large proportion of cases, the secondary B cell malignancies reflected the emergence of immunophenotypically and genetically different clones. An immature type plasma cell myeloma developed in a 73-year-old female patient in whom CLL was diagnosed four years previously. The CLL expressed CD5, CD19, CD23, CD38 and surface kappa light chain, but were negative for ZAP-70. Trisomy 12 was detected by FISH analysis. PCR analysis of the peripheral blood for immunoglobulin heavy chain genes demonstrated two sharp bands that were initially interpreted as biallelic heavy chain gene rearrangements. Myeloma cells were CD38 and CD138 positive, CD19 negative and expressed cytoplasmic kappa light chain, but not heavy chains. In order to investigate the clonal relationship between these B cell malignancies, a detailed analysis of VHDJH and VκJκ gene rearrangements in individually sorted CD5 and CD19 double-positive CLL cells and also in CD38-positive and CD19-negative myeloma cells by single cell PCR of genomic DNA and direct sequencing was carried out. This technique permitted identification and pairing of both the heavy and light chain immunoglobulin genes from the same individually sorted cell. A total of 17 individual CLL and 23 myeloma cells were successfully analyzed. Our analysis demonstrated (a) the presence of two discrete clones of CLL, one with usage of [VH1-2*04/D3-3*01/J3*02]-[Vκ2-28*01/J1*01] without VH and Vκ hypermutation and the other with usage of [VH1-2*04/D4-11*01/J6*02]-[Vκ1-5*03/J1*01] with VH and Vκhypermutation; (b) no clonal relationship between the CLL and myeloma cells that utilized different VHDJH and VκJκ rearrangements [VH3-66*02/3-10*01/J4*03]-[Vκ1-33*01/J2*02] with VH and Vκ hypermutation. To our knowledge, this is the first demonstration of a biclonal CLL with mutated and unmutated clones in the same patient along with a third clonally unrelated B cell malignancy. This result suggests that single cell analysis may be necessary to detect subtle biclonality of CLL that might be associated with a more aggressive phenotype.

Description: An imbalance between cellular apoptosis and survival may be critical for the pathogenesis of lymphoma. Therefore, the gene expression pattern in lymph node preparations from patients with mantle cell lymphoma (MCL) was compared to the pattern in nonmalignant hyperplastic lymph nodes (HLs). Oligonucleotide microarray analysis was performed comparing 5 MCLs to 4 HLs using high-density microarrays. The expression data were analyzed using Genespring software. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction using the RNA extracted from 16 MCL and 12 HL samples. The focus was on 42 genes that were at least 3-fold down-regulated in MCL; in addition to the B-cell leukemia 2 (BCL2) system other apoptotic pathways were altered in MCL. The FAS-associated via death domain (FADD) gene that acts downstream of the FAS cascade as a key gene to induce apoptosis was more than 10-fold down-regulated in MCL. Furthermore, the death-associated protein 6(DAXX) gene, the caspase 2 (CASP2) gene, and the RIPK1 domain containing adapter with death domain(RAIDD) gene, which are key genes in other proapoptotic pathways, were also decreased in the MCL samples. The suggestion is made that in addition to the known overexpression of cyclin D1, which drives entry into the cell cycle, disturbances of pathways associated with apoptosis contribute to the development of MCL.

Description: The circadian system regulates numerous physiological processes including adaptive immune system. Here we show that mice deficient for the circadian genes Cry1 and Cry2, (Cry double knockout [DKO]) display an autoimmune phenotype including higher serum IgG concentration than wild type (WT) mice, presence of serum anti-nuclear antibodies, precipitation of IgG, IgM and complement 3 (C3) in glomeruli, and massive infiltrations of leukocytes into the lung and kidney. A large panel of autoantigens demonstrated that the sera of the Cry DKO mice but not the WT mice, had autoantibodies covering most of the specificities reported to be present in patients with SLE, rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome and other autoimmune disorders. Taken together, lost of the CRY circadian protein leds to severe autoimmunity. Furthermore, flow cytometry analysis of lymphoid organs showed lower pre-B cell numbers and higher mature recirculating B cells in the bone marrow as well as increased number of B2 B cells in the peritoneal cavity of Cry DKO mice. The BCR-proximal signaling pathway plays a critical role in peripheral B cell tolerance and activation. Activation of splenic B cells from the Cry DKO mice elicited markedly enhanced and prolonged tyrosine phosphorylation of cellular proteins compared to WT mice, suggesting that a very active BCR signaling pathway may contribute to impaired B cell tolerance in the Cry DKO mice. In summary, our results suggest that B cell development, as well as the intrinsic checkpoints of immune tolerance, are under direct circadian control. Disclosures No relevant conflicts of interest to declare.

Description: Arsenic trioxide (ATO) induces apoptosis of plasma cells through a number of mechanisms including inhibiting DNA binding by NF-κB. These results suggest that this agent may be synergistic when combined with other active anti-myeloma drugs. To evaluate this we examined the effect of ATO alone and in combination with anti-myeloma treatments evaluated in vitro with MTT assays and using our severe combined immunodeficient (SCID)-hu murine myeloma models. First, we determined the effects of combining ATO with bortezomib or melphalan on the myeloma cell lines RPMI8226 and U266. Cell proliferation assays demonstrated marked synergistic anti-proliferative effects of ATO at concentrations ranging from 5x10−5M – 5x10−9M and melphalan concentrations ranging from 3x10−5M – 3x10−9M. Similar effects were observed when these cell lines were treated with bortezomib and varying concentrations of ATO (5x10−5 M – 5x10−10 M). We also investigated the potential of ATO to increase the efficacy of anti-myeloma therapies in our SCID-hu murine model LAGλ–1 (Yang H et al. Blood 2002). Each SCID mouse was implanted with a 0.5 cm3 LAGλ–1 tumor fragment into the left hind limb muscle. Mice were treated with ATO alone at 6.0 mg/kg, 1.25 mg/kg, 0.25 mg/kg, and 0.05 mg/kg intraperitoneally (IP) daily x5/week starting 19 days post-implantation. Mice receiving the highest dose of ATO (6.0 mg/kg) showed marked inhibition of tumor growth and reduction of paraprotein levels while there was no effect observed in all other treatment groups. Next, 27 days following implantation of our LAGλ–1 intramuscular (IM) tumor, LAGλ–1 mice were treated with ATO (1.25 mg/kg) IP, bortezomib (0.25 mg/kg), or the combination of both drugs at these doses in the schedules outlined above. ATO or bortezomib treatment alone had no anti-myeloma effects at these low doses consistent with our previous results whereas there was a marked decrease in both tumor volume (57%) and paraprotein levels (53%) in mice receiving the combined therapy. The combination of melphalan and ATO was also evaluated in this model. LAGλ–1 bearing mice received therapy with melphalan IP x1/weekly at 12.0 mg/kg, 6.0 mg/kg, 0.6 mg/kg, and 0.06 mg/kg starting 22 days post-implantation and showed no anti-myeloma effects. Twenty-eight days following implantation of LAGλ–1 tumor, mice received ATO (1.25 mg/kg) or melphalan (0.6 mg/kg) alone at doses without anti-myeloma effects, or the combination of these agents at these doses. The animals treated with these drugs alone showed a similar growth and increase in paraprotein levels to control mice whereas the combination of ATO and melphalan at these low doses markedly suppressed the growth of the tumor by 〉50% and significantly reduced serum paraprotein levels. These in vitro and in vivo studies suggest that the addition of ATO to other anti-myeloma agents is likely to result in improved outcomes for patients with drug resistant myeloma. Based on these results, these combinations are now in clinical trials with promising early results for patients with drug resistant myeloma.

Description: Introduction Predicting treatment outcome of acute leukemia has been an important issue. Many factors have been elucidated. We evaluated the impact of donor's availability on treatment outcome including mortality in patients with acute lymphoblastic leukemia (ALL). Methods A total of 294 patients with ALL were evaluated after receiving chemotherapy between year 2001 and July 2014 at our center. Patients were assessed for the need of hematopoietic stem cell transplants (HSCT), availability of HLA sibling match donor and the impact on overall outcome. Indications for transplantation were defined as (1) WBC-〉100,000 for T-ALL, 〉30 for B-ALL with, (2) cytogenetic abnormalities of t(9:22), (4:11) or (1:19), (3) relapse or primary refractory disease or (4) MRD positivity. Patients were divided into 3 categories, group A with an indication for HSCT and available donor (HSCT+/D+), group B with HSCT indication but no available donor (HSCT+/D-) and group C with no indication for HSCT regardless of donor status (HSCT-). Results The median age was 20 (14-63 years). 95 (32%) were female while 198 (68%) were male. 276 (86%) patients were newly diagnosed while 18 (14%) were relapsed. Immuno-phenotype was B for 191 (65%), T for 91 (31%) versus mixed lineage for 12 (4%). 33 (11%) patients were positive for Philadelphia chromosome. Median WBC at diagnosis was 23.2X109/L CNS involvement was positive in 26 (8%) patients. With a median follow-up of 60 months for survivors (range 2 to 116.5 months), 148 (50%) patients showed HSCT+/D+, HSCT+/D- in 79 (27%) and HSCT- in 67 (23%) patients. the 5-year OS for HSCT+/D-, HSCT+/D+ and HSCT- were 29%, 57% and 55% (p value

Description: Metastatic disease of the bone is a rare complication of chronic lymphocytic leukemia (CLL), it may be result from richter's transformation or metastatic from non lymphoid malignancies. CLL is the most common form of adult leukemia, with the median age of 70 years at diagnosis [Siegel et al. 2013]. The diagnosis is established by blood counts, blood smears, and immunophenotyping of circulating B-lymphocytes.The result is the increased number of lymphocytes in the peripheral blood, leukocytosis with absolute lymphocytosis, the increase of the lymphnodes, the increase in size of the spleen. The diagnosis of chronic lymphocytic leukemia B requires the presence of Clonal B cells in the peripheral blood at or above 5,000 / ul for at least 3 months. Typing immunophenotypical pathological lymphocytes are positive for surface antigens CD5, CD19, CD23, weakly positive for CD20 and CD22, generally negative FMC7 and CD79b; also expressing surface immunoglobulins. The Rai and Binet staging systems, which are established by physical examination and blood counts, have been recognized as standards for deciding whether to begin treatment. Patients with active or symptomatic disease or with advanced Binet or Rai stages require therapy. For fit patients, chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab represents the current standard therapy. For unfit patients, treatment with an anti-CD20 antibody (obinutuzumab, rituximab, ofatumumab) plus a milder chemotherapy (Chlorambucil) may be applied. At relapse, if the treatment-free interval exceeds two to three years, the initial treatment may be repeated, if the disease relapses earlier, drugs such as bendamustine (plus rituximab), alemtuzumab, lenalidomide, ofatumumab, ibrutinib, or idelalisib, must be choosen. Patients with a del(17p) or TP53 mutation can be treated with ibrutinib or a combination of idelalisib and rituximab. in relapsing patients with TP53 mutations or del(17p) or patients that are refractory to repeated chemoimmunotherapies, an allogeneic SCT may be considered [Hallek M 2015]. In this article we show a case of a 66-year-old man with CLL and a bone localization. In 2011 diagnosis of CLL, Rai Stage 0, Binet Stage A. Principal characteristics at diagnosis: HB 13.2 g /dl, White Blood Cells 15.800 / mm3, lymphocytes 61%, neutrophils 32%, monocytes 4%, platelets 141.000/mm3; normal hepatic end renal function; flowcytometric immunophenotyping of the peripheral blood revealed B-cell CLL; prognostic factors: CD38 negative, ZAP70 positive, rearrangement of the immunoglobulins mutated; FISH: negative; CT chest / abdomen / pelvis: presence of multiple aorto-pulmonary and axillary adenopathies (max diameter of 2 centimeters); bone marrow biopsy: infiltration of CLL equal to 60% of global cellularity. The patient was only observed until January 2015, when he was hospitalized due to acute anemia, requiring supportive therapy, and right foot pain . So it was decided to re-evaluate the whole disease in order to decide whether to start chemotherapy. The disease was staged again with instrumental and laboratory tests: presence of renal insufficiency, egd and colonoscopy negative, Coombs' test negative, bone marrow biopsy confirmed the diagnosis of chronic lymphocytic with bone marrow infiltration of 90%, abdomen ultrasound showed only moderate splenomegaly. On February, persistence of right foot pain and appearance of swelling, assessed by the orthopedic as a suspected algic and dystrophic syndrome. So he suggested to perform scintigraphy which revealed: pronounced inflammatory osteometabolic reaction of the right tibia/fibula/ankle third distal which could be referred, in the first evaluation, to algic and dystrophic syndrome. However, a local biopsy was performed: localization of chronic lymphocytic leukemia. On March 2015 a total body TC showed 2 nodular calcifications in the right lung lobe, multiple right paratracheal, barety space, aortopulmonary and axillary adenopathies. Prostate size increased. In order to study carefully the liver and prostate lesions, an ultrasound abdomen was performed that documented only enlarged spleen, normal size liver, free of focal disease, increased prostate due to symmetric bilobate hypertrophy . After the second cycle of chemotherapy, prolonged thrombocytopenia, so he continues only with a radiotherapy program. Disclosures No relevant conflicts of interest to declare.