ALBERT

Description: Background/Patients and Methods: NST is increasingly being used as a means of establishing a graft-versus-malignancy (GVM) effect with less regimen related toxicity. Between 9/01 and 7/04, 39 patients (pts) with high risk/relapsed/refractory HM who were not candidates for full intensity allogeneic stem cell transplantation underwent NST using Pentostatin/TBI. The median age of pts was 52 years (range 22–70). The median number of prior therapies was 4 (range 0–8) including prior autologous stem cell transplantation in 22 pts. Diseases transplanted included chronic lymphocytic leukemia/indolent non-Hodgkin’s lymphoma (NHL, n=6), aggressive NHL (n=8), mantle cell lymphoma (n=3), Hodgkin’s disease (n=6), myeloproliferative disorders (n=4), myelodysplastic syndromes (n=4), and acute myelogenous leukemia (AML, n=8). Conditioning consisted of Pentostatin 4 mg/m2 daily on day −21, −20, and −19, followed by 200 cGy TBI on day −1. Post-grafting immunosuppression consisted of cyclosporine/mycophenolate mofetil. Results: Stem cell transplantation was from matched related (n=14) or unrelated (n=25) donors. Death prior to 100 days post transplant occurred in 7 patients. Grade III/IV toxicities included hematologic (n=10 pts), infectious (n=5) and other non-infectious (n=4). The median nadir values (day −21 to day 0) for hemoglobin, neutrophil count and platelet count were 10.7 g/dl (range 7.8–12), 1056/mm3 (range 0–5336), and 174/mm3 (range 24–523) respectively. Three pts failed to engraft; two patients with myelofibrosis (both of whom had autologous reconstitution) and one patient with high risk AML (who died of complications of fungal sepsis without hematologic recovery). The median chimerism values for CD3+ cells and WBC at day 28 are 80% and 95% donor cells respectively. The median chimerism values for CD3+ and WBC at day 70 are 95% and 95% respectively. There have been no late graft failures. The cumulative incidence of all grades of acute graft-versus-host disease at day 100 was 40% and was more common in unrelated donor transplants (60% vs. 15%, P=0.012). Chronic graft-versus-host disease has developed in 69% of patients. The cumulative incidence of relapse for all patients is 30%, and is lower for unrelated donor transplants than matched related donor transplants (46% vs. 20%, P=0.02). The probability of event-free and overall survival at two years is 52% and 56% respectively. Conclusions: This regimen is associated with acceptable toxicity. Engraftment has not been an issue with the exception of two pts with myelofibrosis. Pts receiving unrelated donor grafts have a higher incidence of graft-versus-host disease and a lower relapse rate. This represents indirect support for the presence of a GVM effect. A prospective study using a modified Pentostatin schedule (starting at day − 10) is ongoing based on the nadir of host T-cells identified in this study.

Description: Hematopoietic stem cell (HSC) quiescence is crucial for maintaining lifelong blood production and preventing potentially toxic overproduction of blood cells. HSC are capable of re-entering quiescence following exposure to pro-inflammatory stimuli such as interleukin (IL)-1, implying the existence of one or more 'braking' mechanisms that limit and/or overcome the mitogenic properties of these signals to limit HSC cell cycle entry. However, mechanism(s) regulating HSC quiescence during chronic inflammation have yet to be fully elucidated. In the present study, we find that the master myeloid transcription factor PU.1 represses Myc-regulated cell cycle and protein synthesis pathways in HSC during chronic inflammation, thereby guarding HSC quiescence in this context. To gain insight into HSC cell cycle regulation in the context of chronic inflammatory signaling, we performed cell cycle and RNA-seq analysis on purified HSC from mice injected daily for 20 days with IL-1β. Strikingly, HSC from IL-1-treated mice remained in a quiescent state, and our RNA-seq analyses uncovered a significant decrease in mRNA transcripts from pathways related to cellular proliferation, translation, and ribosome biogenesis. Also, flow cytometry analyses revealed that IL-1 significantly reduced Myc protein expression and ribosomal protein S6 phosphorylation in HSC. Altogether, these data identify an inflammation-induced cell cycle restriction gene program that limits HSC proliferation in response to inflammation. Strikingly, ingenuity pathway analysis (IPA) predicted PU.1as a possible driver of the cell cycle restriction gene program. Indeed, PU.1 mRNA and protein levels in HSC increased significantly in PU.1-eYFP knockin reporter mice treated with IL-1. Furthermore, fractionating HSC based on reporter levels showed that IL-1-exposed HSC expressing high PU.1 levels activated the cell cycle restriction program more robustly than HSC with lower PU.1 levels or from untreated control mice. Along these lines, cell cycle analyses showed that PU.1-high HSC were quiescent following IL-1 exposure. Surprisingly, functional assays revealed that the PU.1-high fraction became enriched for functional HSC following IL-1 exposure, in contrast to a primarily PU.1-low phenotype under control conditions. Collectively, these data show that low Myc levels, a quiescent cell cycle state and functional HSC identity are all associated with high PU.1 levels during chronic inflammation. To address the requirement for PU.1, we compared HSC following IL-1 exposure from wild-type (WT) and PU.1-knockin (KI) mice, which express ~30% of normal PU.1 levels due to a point mutation in the PU.1 upstream regulatory element (URE). Strikingly, IL-1 induced aberrant HSC expansion in the BM of PU.1 KI mice, underwritten by IL-1-induced quiescence loss in PU.1 KI HSC. Excess cell cycle activity in HSC from IL-1-treated PU.1 KI mice was associated with Myc overexpression, hyperinduction of cycle and protein synthesis genes, and an increased protein synthesis rate. Interestingly, IL-1-treated PU.1 KI mice also exhibited exuberant platelet production and aberrant activation of megakaryocyte (Mk) lineage programs in HSC. Taken together, these data show PU.1 is required to limit HSC proliferation and cell cycle activity, and that failure to upregulate PU.1 during chronic inflammation leads to aberrant HSC expansion and dysregulated Mk lineage output. Altogether, our data identify PU.1 as a key enforcer of HSC dormancy, and as a regulator of HSC proliferation and proteostasis during chronic inflammation. As PU.1 has been shown to slow cell cycle entry to promote myeloid differentiation in actively proliferating cells, our results suggest a mechanistic conservation in which inflammation induces PU.1 expression and engages a similar cell cycle restriction program that preserves HSC quiescence. Such a mechanism can prevent excessive Mk lineage output leading to thrombosis, and/or HSC exhaustion. Importantly, these findings re-cast inflammation-induced PU.1 expression as a guardian of blood system function, rather than a pathogenic by-product. As PU.1 serves a tumor suppressor role, these findings also may provide insight into the link between inflammation and leukemogenesis, particularly in contexts where PU.1 function is impaired in leukemic stem cells (LSC) due to mutation or indirect mechanism(s). Disclosures No relevant conflicts of interest to declare.

Description: Key Points PRS, based on the known CLL loci, predicts CLL risk with high discrimination. This PRS predicts risk of monoclonal B-cell lymphocytosis, a precursor to CLL and a condition that has clinical impact beyond risk for CLL.

Description: The multimeric size and the function of circulating von Willebrand factor are modulated via its proteolytic cleavage by the plasma metalloproteinase, ADAMTS13. It is unclear how ADAMTS13 activity is regulated within the vascular system. In the absence of a regulatory mechanism, ADAMTS13 activity might compromise platelet adhesion at sites of vascular injury. We hypothesized that at sites of vascular injury, ADAMTS13 activity could be regulated locally by coagulation proteinases. Initiation of coagulation in human plasma resulted in the disappearance of added full-length recombinant ADAMTS13. This loss was inhibited by hirudin. Using purified proteins, we showed that ADAMTS13 is proteolyzed at several cleavage sites by thrombin in a time- and concentration-dependent manner. Furthermore, this proteolysis ablated ADAMTS13 activity against purified von Willebrand factor. Preincubation of thrombin with soluble thrombomodulin, but not heparin, inhibited the proteolysis of ADAMTS13, suggesting the involvement of thrombin exosite I (and not exosite II) in ADAMTS13 recognition. Plasmin also cleaved ADAMTS13 into similar fragments, resulting in the loss of ADAMTS13 activity. This study demonstrates the susceptibility of ADAMTS13 to proteolytic inactivation and suggests possible roles for thrombin and plasmin at sites of vascular injury.

Description: Recently we have identified subgroups of de novo primary diffuse large B-cell lymphoma (DLBCL) based on complementary DNA microarray-generated gene expression profiles. To correlate the gene expression profiles with cytogenetic abnormalities in these DLBCLs, we examined the occurrence of the t(14;18)(q32;q21) in the 2 distinctive subgroups of DLBCL: one with the germinal center B-cell gene expression signature and the other with the activated B cell–like gene expression signature. The t(14;18) was detected in 7 of 35 cases (20%). All 7 t(14;18)-positive cases had a germinal center B-cell gene expression profile, representing 35% of the cases in this subgroup, and 6 of these 7 cases had very similar gene expression profiles. The expression of bcl-2 and bcl-6 proteins was not significantly different between the t(14;18)-positive and -negative cases, whereas CD10 was detected only in the group with the germinal center B-cell expression profile, and CD10 was most frequently expressed in the t(14;18)-positive cases. This study supports the validity of subdividing DLBCL into 2 major subgroups by gene expression profiling, with the t(14;18) being an important event in the pathogenesis of a subset of DLBCL arising from germinal center B cells. CD10 protein expression is useful in identifying cases of DLBCL with a germinal center B-cell gene expression profile and is often expressed in cases with the t(14;18).

Description: Gastrointestinal (GI) graft-versus-host disease (GvHD) is a major barrier in allogeneic hematopoietic stem-cell transplantation (AHST). The metabolite retinoic acid (RA) potentiates GI-GvHD in mice via alloreactive T-cells expressing the RA-receptor-alpha (RARα), but the role of RA-responsive cells in human GI-GvHD remains undefined. We therefore used conventional and novel sequential immunostaining and flow cytometry to scrutinize RA-responsive T-cells in tissues and blood of AHST patients and characterize the impact of RA on human T-cell alloresponses. Expression of RARα by human mononuclear cells was increased after RA exposure. RARαhi mononuclear cells were increased in GI-GvHD tissue, contained more cellular RA-binding proteins, localized with tissue damage and correlated with GvHD severity and mortality. Using a targeted candidate protein approach we predicted the phenotype of RA-responsive T-cells in the context of increased microenvironmental IL-23. Sequential immunostaining confirmed the presence of a population of RARahi CD8 T-cells with the predicted phenotype, co-expressing the effector T-cell transcription factor T-bet and the IL-23-specific receptor. These cells were increased in GI- but not skin-GvHD tissues and were also selectively expanded in GI-GvHD patient blood. Finally, functional approaches demonstrated RA predominantly increased alloreactive GI-tropic RARahi CD8 effector T-cells, including cells with the phenotype identified in vivo. IL-23-rich conditions potentiated this effect by selectively increasing b7 integrin expression on CD8 effector T-cells and reducing CD4 T-cells with a regulatory cell phenotype. In conclusion we have identified a population of RA-responsive effector T-cells with a distinctive phenotype which are selectively expanded in human GI-GvHD and represent a potential new therapeutic target.

Description: Myeloid derived suppressor cells (MDSCs) have been reported to be expanded in cancer patients, following growth factor administration and after chemotherapy. These cells have been associated with a loss of T-cell number and function and provide one mechanism of immune evasion. We examined the effect of dose dense chemotherapy on immune phenotypes and function in patients with breast cancers 4 cms or larger and/or four or more involved nodes. The adjuvant therapy was dose-dense doxorubicin, cyclophosphamide (AC) followed by paclitaxel (P), then 33 doses of radiation (R). Blood samples were obtained and studied prior to therapy, 1 week post AC and 1, 15 and 21 weeks post P and then 3, 6 and 12 months later. Flow cytometric analyses of cellular phenotypes were done on these blood specimens and compared to the levels prior to therapeutic intervention and to normal age and sex matched donors. Twenty-three pts have been followed a median of 29 months (range 5.5–50.5 months) from study entry. Two patients relapsed 8 and 23 months after diagnosis. T-cell CD-4 numbers declined following AC from an average of 4.9±0.5 ×106/ml to 1.7±0.3×106/ml, but increased to an average of 2.7± 0.3 × 106/ml, 21 weeks after P or 12 weeks after R. In this study the MDSCs were defined as Lin- (CD3, CD19, CD14 and CD13), HLA-DR- and CD33+. The numbers of MDSCs, which in normal donors were 0.62±0.16×106/ml and in the cancer patients at diagnosis were 11.8±9.6×106/ml increased to 58.4±25.9×106/ml 15 weeks after R. They remained significantly elevated through one year after diagnosis when they were 27.3±12.3×106/ml. The majority of the MDSCs had a high side scatter and forward scatter by flow analysis suggesting a granulocytic commitment rather than a monocytic commitment. The increase in MDSC numbers was apparently associated with R as the numbers of MDSCs were not significantly increased by AC (15.7±13.5×106/ml) or P (10.9±6×106/ml) one week following completion of each cycle of dose dense therapy. In association with the increase in MDSCs there was a significant decrease in PHA proliferation by the peripherial blood mononuclear cells (MNCs) and suppressive activity by irradiated MNC for allergenic lymphocytes.

Description: Background: Shwachman-Diamond Syndrome (SDS) is a bone marrow failure disorder caused by impaired removal of EIF6 from the nascent 60S ribosome subunit, resulting in defective ribosome assembly. SDS patients have a high risk of myeloid neoplasms (MN) and the prognosis of those that develop MN is poor. Knowledge of the kinetics and functional consequences of somatic mutation acquisition in SDS may offer insight into mechanism of transformation and the potential for therapuetic intervention. Methods: We performed whole exome sequencing of 45 samples from 30 patients, and validated recurrent somatically mutated genes using targeted sequencing with error suppression in prospectively collected samples from 110 patients in the North American SDS Registry. We correlated mutation status with clinical outcome and performed functional studies to understand the consequence of somatic mutations in SDS. Results: We detected somatic mutations in 74 of 98 (76%) patients with germline biallelic SBDS mutations (median 2 mutations/patient, range 0-21). We found no mutations in patients with SDS-like disease; those who have clinical features of SDS without disease defining mutations. Of the 83 patients with SDS without a MN diagnosis, 60 (72%) had detectable clonal hematopoiesis (CH), 40 of whom had more than one mutation (median 3, range 1-21). The most frequently mutated genes were EIF6 (60/98, 61%),TP53 (44/98, 45%), PRPF8 (12/98, 12%), and CSNK1A1 (6/98, 6%). Among SDS patients with TP53 mutated CH, 90.9% (30 of 33) had concurrent EIF6 mutations. To determine whether EIF6 and TP53 mutations occur in the same or different clones, we performed single cell DNA sequencing. Among the 47 clones identified with either EIF6 or TP53 mutations, 24 had a sole EIF6 mutation, and 21 had a sole TP53 mutation, showing that these mutations arise in separate clones. To study the functional consequences of EIF6 missense mutations, we cloned 7 patient-derived mutations and generated cell lines expressing wild-type or mutant EIF6 cDNA. We found six mutants (I13N, R67W, G69S, P73R, A194T, G196R) reduced levels of EIF6 protein compared with wild type EIF6, despite comparable abundance of mRNA. The most common recurrent mutation, N106S, was found in 20% of patients and, by contrast to others listed above, did not change protein expression. This mutation is located at the EIF6/60S protein interface and disrupted the interaction of N106S-EIF6 with the 60S subunit as measured by polysome profiling followed by western blotting. To compare the effects of EIF6 versus TP53 somatic mutations in context of SDS deficient translation, we measured ribosome maturation and translation in SDS cells containing shRNAs targeting EIF6 or TP53. EIF6 knockdown ameliorated the SDS defect, reflected by improved ribosome joining (normalization of the 80:60s ratio) and enhanced protein translation (increased O-propargyl-puromycin incorporation), whereas TP53 knockdown had no effect. Knockdown of EIF6 in SDS deficient cells decreased p53 pathway activation as demonstrated by decreased CDKN1A expression. TP53 mutations were significantly associated with MN diagnosis (p=0.023), but were also common in SDS CH and typically stable over time. To identify the characteristics associated with transformation, we analyzed exomes from 7 patients with TP53 mutated myeloid malignancy for allelic imbalances at the TP53 locus and found that all 7 had biallelic alteration of TP53. Using single cell DNA sequencing from serial samples, we observed that TP53 LOH can precede transformation by several years and can distinguish pre-leukemic clones from indolent clones with monoallelic TP53 alterations. Somatic EIF6 mutations were not found in the leukemic clones. These results suggest early detection of TP53 LOH may distinguish clones with leukemic potential. Conclusions: In SDS, impairment of ribosome maturation drives selection of clones with somatic EIF6 or TP53 mutations. EIF6 mutations promote competitive fitness by rescuing the SDS ribosome defect and decreasing p53 pathway activation, and do not contribute to malignant transformation. TP53 mutations decrease checkpoint activation without affecting ribosome assembly. These results provide genetic evidence that germline SBDS deficiency causes a global, disease-specific HSC fitness constraint that drives parallel development of somatic CH and provides a mechanistic rationale for clinical surveillance. Disclosures Dale: Emendo BioTherapeutics: Consultancy; X4 Pharmaceuticals: Research Funding; X4 Pharmaceuticals: Honoraria. Gansner:Alnylam Pharmaceuticals: Current Employment, Current equity holder in private company. Edwards:Jazz Pharmaceuticals: Consultancy, Honoraria. Fleming:DISC Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lindsley:MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy; Bluebird Bio: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding.

Description: Loss-of-function mutations in KMT2D are a striking feature of the germinal centre (GC) lymphomas, resulting in decreased H3K4-methylation and altered gene expression. We hypothesised that inhibition of the KDM5 family, which demethylates H3K4me3/me2, would re-establish H3K4-methylation and restore the expression of genes repressed upon loss of KMT2D. KDM5-inhibition increased H3K4me3 levels and caused an anti-proliferative response in vitro, which was markedly greater in both endogenous and CRISPR-edited KMT2D mutant DLBCL cell lines, while tumour growth was inhibited in KMT2D mutant xenografts in vivo. KDM5-inhibition reactivated both KMT2D-dependent and -independent genes, resulting in diminished B-cell signalling and altered expression of BCL2 family members, including BCL2 itself. KDM5-inhibition may offer an effective therapeutic strategy for ameliorating KMT2D loss-of-function mutations in GC-lymphomas.

Description: We have treated a total of 30 patients with autologous T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19; 22 of 27 evaluable patients obtained either complete remissions (CR) or partial remissions (PR). Ten patients remain in ongoing CRs of 1 to 37 months duration. The CAR was encoded by a gammaretroviral vector and included the variable regions of an anti-CD19 antibody along with CD28 and CD3-zeta moieties. The first 21 patients treated on this protocol have been reported (Kochenderfer et al. Blood 2010, Blood 2012, and Journal of Clinical Oncology 2014). To enhance the activity of the transferred CAR T cells, T-cell infusions in the previously reported patients were preceded by a chemotherapy regimen of high-dose cyclophosphamide (60-120 mg/kg) plus fludarabine. In an attempt to reduce the overall toxicity of our anti-CD19 CAR treatment protocol, we substantially reduced the doses of chemotherapy administered before CAR T-cell infusions. This abstract communicates results from 9 patients with B-cell lymphoma who received a single infusion of 1x106 anti-CD19-CAR-expressing T cells/kg bodyweight preceded by a low-dose chemotherapy regimen consisting of cyclophosphamide 300 mg/m2 and fludarabine 30 mg/m2 (Table). Each chemotherapy agent was administered daily for 3 days. Eight of the 9 treated patients had DLBCL (diffuse large B-cell lymphoma) that was refractory to chemotherapy (chemo-refractory) or that had relapsed less than 1 year after autologous stem cell transplantation (ASCT). Both of these clinical situations carry a grim prognosis, with median overall survivals of only a few months. Despite the very poor prognoses of our patients, one patient with DLBCL obtained a CR and 4 DLBCL patients obtained PRs. In some patients, PRs included resolution of large lymphoma masses. Compared to our previous experience with anti-CD19 CAR T cells preceded by high-dose chemotherapy, toxicity was reduced when CAR T cells were infused after low-dose chemotherapy. None of the 9 patients treated with low-dose chemotherapy and CAR T cells required vasopressor drugs or mechanical ventilation, although some patients did have short-term neurological toxicity. Cytopenias were mild with a mean of only 1.4 days of blood neutrophils