ALBERT

Description: Introduction Mutant NRAS and KRAS lead to the activation of the RAS/RAF/MEK/ERK pathway in approximately 50% of multiple myeloma (MM). Blocking this pathway with MEK1/2 inhibitors (MEKi) such as trametinib (Tram) is a therapeutic option but the response rate in MM varies between 30-50% (Heuck et al, Leukemia 2015). In MM it is unknown whether RAS mutation status correlates with sensitivity to Tram. The purpose of this study was to characterize factors which predict response to Tram and to identify mechanism mediating resistance. Methods We established the IC50 of Tram using MTT assays in 32 MM cell lines (HMCL) including 16 RAS mutant positive (RASm+), and 15 wildtype RAS (RASm-), and 1 BRAF mutant (BRAFm+) line which acted as a positive control. HMCLs were classified according to the IC50 value as sensitive (10μM). All lines underwent immunoblotting for pERK at baseline and following treatment with serial concentrations of Tram to identify correlation of activation with sensitivity. BrdU incorporation analyzed by FACS was performed to determine the molecular action of Tram. A lentiviral mediated expression system was used to engineer a MAF overexpressing cell line in a RASm+ HMCL lacking MAF (MMRASm+MAF) and silencing MAF in two lines with co-occurring MAF and RASm+ (MMRASm+shMAF). The clinical characteristics of 84 relapsed RASm+ patients who received Tram either as a single agent or in combination with other anti-MM therapies were also examined. Results 6/16 (37.5%) of RASm+ HMCLs were sensitive to Tram, 5/16 (31.1%) were IMS and 5/16 (31.1%) resistant. There was no difference in sensitivity to Tram between KRASm+ (IC50 = 9.5μM, n = 11) and NRASm+ (IC50 =12.5μM, n=4, p=0.65). In contrast, 13/15 (87%) RASm- HMCLs were resistant to Tram. Mechanistically, Tram blocked cell cycle progression in Tram-sensitive RASm+ cells with an increase in G0/G1 phase (22.25%) and a decrease in S phase (16.76%) compared with untreated controls (p

Description: Key Points Immune responses to FVIII sequence variants encoded by ns-SNPs do not contribute appreciably to inhibitor development in African Americans. African American HA subjects with an intron-22 inversion had a 2- to 3-times-higher inhibitor incidence than whites with the same mutation.

Description: Background: Activating mutations of NRAS and KRAS genes are common in newly diagnosed acute myeloid leukemia (AML), occurring in 11-16% and 4-5% of patients, respectively. RAS mutations are frequently acquired at time of progression from MDS to AML and are associated with poor survival. Next generation sequencing (NGS) at diagnosis and during complete remission has shown that RAS mutations have high clearance rates with induction chemotherapy. In the CALGB 8525 study, RAS-mutant younger patients (age

Description: Multiple myeloma (MM) is characterized by osteolytic bone lesions (OBL) that arise as a consequence of osteoblast inactivation and osteoclast activation adjacent to tumor foci within bone. Wnt signaling in osteoblasts regulates osteoclastogenesis through the differential activation and inactivation of Receptor Activator of Nuclear factor Kappa B Ligand (RANKL) and osteoprotegerin (OPG), positive and negative regulators of osteoclast differentiation, respectively. We demonstrate here that MM cell–derived DKK1, a soluble inhibitor of canonical Wnt signaling, disrupted Wnt3a-regulated OPG and RANKL expression in osteoblasts. Confirmed in multiple independent assays, we show that pretreatment with rDKK1 completely abolished Wnt3a-induced OPG mRNA and protein production by mouse and human osteoblasts. In addition, we show that Wnt3a-induced OPG expression was diminished in osteoblasts cocultured with a DKK1-expressing MM cell line or primary MM cells. Finally, we show that bone marrow sera from 21 MM patients significantly suppressed Wnt3a-induced OPG expression and enhanced RANKL expression in osteoblasts in a DKK1-dependent manner. These results suggest that DKK1 may play a key role in the development of MM-associated OBL by directly interrupting Wnt-regulated differentiation of osteoblasts and indirectly increasing osteoclastogenesis via a DKK1-mediated increase in RANKL-to-OPG ratios.

Description: Juvenile and chronic myelomonocytic leukemias (JMML and CMML) are aggressive myeloid malignancies categorized as myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). Chemotherapy has little benefit for MDS/MPN patients, and new therapies are needed. We have used mouse models investigate the potential of signal transduction inhibitors in MDS/MPN, as JMML and CMML are associated with mutations in NRAS, KRAS, PTPN11, CBL, or NF1 that activate Ras signaling. Conditional Mx1-Cre, KrasLSL-D12 (designated KrasD12) mice develop an aggressive and fully penetrant MDS/MPN characterized by leukocytosis, splenomegaly, anemia, and death by 10-16 weeks of age. Mx1-Cre, Nf1flox/- mice (hereafter Nf1Δ/-) undergo conditional loss of Nf1. These mice also develop MDS/MPN, but the disease is more indolent. We and others have investigated inhibition of effector networks downstream of Ras, such as the Raf/MEK/ERK (MAPK) and phosphotidylinositol-3 kinase (PI3K)/Akt pathways. We previously showed that the MEK inhibitor PD0325901 induced sustained hematologic improvement in both KrasD12 and Nf1Δ/- mice. We also have reported that the class I PI3K inhibitor GDC-0941 improves hematologic function and prolongs survival in KrasD12 mice. However, GDC-0941 and other PI3K inhibitors attenuate both PI3K/Akt and Raf/MEK/ERK pathways due to effects of PI3K upstream of Ras. Therefore, the benefit from GDC-0941 could have been due to its modulation of Raf/MEK/ERK signaling. Here, we specifically test the importance of Akt signaling in MDS/MPN in KrasD12 and Nf1 mouse models using the allosteric inhibitor MK-2206. This compound binds to the interface of the PH and kinase domains of Akt1, Akt2, and Akt3, and does not inhibit any of 250 other kinases at 1 µM. MK-2206 induced substantial improvement in both KrasD12 and Nf1Δ/- mice. Mice treated with MK-2206 had pronounced reduction in leukocytosis, reticulocytosis and splenomegaly, increased hemoglobin concentration, and prolonged survival. MK-2206 had no hematologic effects in control WT mice, indicating some selectivity against aberrant hematopoiesis. Importantly, MK-2206 inhibited Akt but not Raf/MEK/ERK or Jak/STAT signaling. This demonstrates that canonical PI3K/Akt signaling plays an important role in Ras-driven MDS/MPN. Furthermore, combined inhibition of MEK and Akt with PD0325901+MK-2206 yielded a greater improvement in splenomegaly than either agent alone in both KrasD12 and Nf1Δ/- models. Akt has multiple effectors relevant to hematopoiesis and leukemia. Of these, mTOR is of particular interest for targeted cancer therapy. Therefore, we tested the response of KrasD12 mice to rapamycin, a partial inhibitor of mTOR with preferential activity against the mTORC1 complex. KrasD12 mice demonstrated variable responses to rapamycin, with approximately half undergoing a complete and durable hematologic response and the remainder having no response. Together, these studies further implicate PI3K/Akt signaling as a pathogenic effector downstream of Ras in MDS/MPN and support the idea that inhibitors targeting this pathway may have a role in treatment of JMML or CMML. Disclosures No relevant conflicts of interest to declare.

Description: Background Measurable residual disease (MRD) is associated with inferior outcomes in patients with acute myeloid leukemia (AML). MRD monitoring enhances risk stratification and may guide therapeutic intervention. Post-induction MRD is frequently cleared with further therapy and the clearance may lead to better outcomes. In contrast, persistent MRD is associated with poor outcomes. At present it is not possible to predict which patients are likely to clear MRD with further therapy. Here we report a simple, objective, widely applicable and quantitative MFC approach using the ratio of blast/PDC to predict persistent MRD and poor outcomes in AML. Patients and Methods A cohort of 136 adult patients with a confirmed diagnosis of AML by WHO criteria who underwent standard induction therapy at a single center between 4/2014 and 9/2017 was initially included. 69 patients achieved complete morphologic remission (36 MRD-neg. and 33 MRD-pos.). MRD status was assessed by MFC using a different from normal (DfN) approach. PDC were quantified as the percent of total WBC by flow cytometry based on low side scatter, moderate CD45, CD303, bright CD123 and HLA-DR expression. Results The proportion of PDC was markedly decreased in patients with AML (≥20% blasts) (N=136) with a median of 0.016% (interquartile range IQR: 0.0019%-0.071%, Figure 1A), more than 10-fold lower than observed in normal controls (median 0.23%, IQR 0.17%-0.34%) (N=20). While there was no difference between MRD-neg. and normal control groups (median 0.31%, IQR: 0.17%-0.49%; vs. 0.28%, IQR: 0.17%-0.34%), MRD-pos. group had significantly reduced PDC proportion compared to the control (median 0.074%, IQR: 0.022%-0.33%, Wilcoxon rank sum, p=0.019). In an attempt to achieve better separation and to eliminate possible effects of hemodilution, the ratio of blast/PDC was calculated by using the proportions of blasts and PDCs out of total WBCs as quantitated by flow cytometry. A cut-off threshold of the blast/PDC ratio of 10 was chosen to separate each group (Figure 1B). Importantly, a ratio cut-off of 10 had a corresponding specificity of 97.4% for predicting MRD positivity status. MRD positivity was significantly associated with inferior overall survival (OS) and relapse-free survival (RFS) in our study cohort (OS HR 4.11 (95% CI: 1.30-13.03), p=0.016; RFS HR 4.20 (95% CI: 1.49-11.82), p=0.007, Figure 1C and D). The 2-year cumulative incidence of relapse in the MRD-neg. group compared to MRD-pos. group was 10% (95% CI: 2-24%) vs. 37% (95% CI: 18-56%, p=0.014). Importantly, blast/PDC ratio ≥10 was also strongly associated with inferior OS and RFS (OS HR 3.12 (95% CI: 1.13-8.60), p= 0.028; RFS HR 4.05 (95% CI: 1.63-10.11), p=0.003, Figure 1E and F), which is similar in magnitude to MRD positivity. Furthermore, MRD-pos. patients with blast/PDC ratio

Description: Background: Many prognostic models have been proposed for staging patients with MM, most recently the ISS system utilizing B2M and albumin. Patients and Methods: In the context of Total Therapy 2 (TT2) for newly diagnosed MM, we identified 220 patients in whom 4 standard prognostic factors (SPF: B2M, albumin, LDH, hemoglobin), imaging data (MRI-defined focal lesions [MRI-FL]), metaphase-derived cytogenetic abnormalities (CA), FISH-derived amp1q21 and del13q14, and gene expression profiling (GEP)-derived data are available. The baseline characteristics and clinical outcome of the 220 patients are similar to those of the entire population of 668 patients receiving TT2. The median follow-up of the 220 patients is 42mo compared to 52mo for all 668 patients. Five multivariate analysis-based prognostic models were derived, utilizing SPF only (model I), with progressive addition of CA (model II), MRI (model III), FISH (model IV) and GEP (model V). Results: In model I, B2M, LDH and hemoglobin, but not albumin were independently significantly associated with overall survival (OS) (R2=22.5%); upon introduction of CA (model II) (p=.008), only LDH remained significant (p=.033) (R2=28.3%); addition of MRI (model III) was not significant (R2=31.1%); addition of FISH (model IV) was significant for amp1q21 (p=.003) but not for del13q13, with CA remaining significant (p=.023) (R2=38.1%); when GEP was added (model V), the 70 gene-derived model (p

Description: Introduction Chromosome instability (CIN) is a driver of copy number aberrations (CNAs) in cancer, and is a major factor leading to tumor heterogeneity and resistance to therapy. By definition, CIN is an increased rate or ongoing acquisition and accumulation of CNAs and not simply the existence of structurally and numerically abnormal aneuploid clones. In multiple myeloma (MM), the most common whole-chromosome CNAs involve either hyperdiploid or non-hyperdiploid clones. Secondary segmental CNAs are associated with high-risk (HR) in MM and involve gains of 1q21 and deletions of 17p (del17p). These types of intra-chromosomal segmental CNAs are also found in the CIN phenotypes of the autosomal recessive (AR) chromosome instability syndromes. These syndromes include Fanconi anemia, Bloom's syndrome, and ICF syndrome (Immunodeficiency, Centromeric instability and Facial anomalies). These chromosome instability syndromes display a spectrum of aberrations characterized by higher rates of chromosomal breaks, chromatid exchanges, quadriradials, and pericentromeric aberrations. In particular, patients with ICF syndrome show a marked increase of 1q12 pericentromeric instability including 1q12 decondensation, triradials, multibranched chromosomes 1q, and 1q micronuclei. ICF patients also show transient 1q aberrations including isochromosome 1q (iso1q) and unbalanced translocations of 1q to 9q and 16q. In MM, we have previously reported increasing pericentromeric instability during tumor progression resulting in increasing CNAs of 1q21 by unbalanced jumping translocations of 1q12 (JT1q12). Strikingly, in a subset of MM patients with 1q21 CNAs of ≥ 5 a distinct cytogenetic phenotype emerges which demonstrates transient 1q12 aberrations including 1q12 decondensation, triradials, and multibranched chromosomes 1q morphologically identical to those seen in ICF patients. In MM this chromosome instability leads to a cascade of increasing clonal 1q21 duplications, iso 1qs, and unbalanced 1q translocations with 16q and 17p, resulting in losses in these receptor chromosomes (RC) and massive intra-clonal CNA heterogeneity. Methods To investigate the cytogenetic impact and progression of high CNAs of 1q21, we performed a comprehensive metaphase analysis of 50 patients showing segmental aneuploidies with 4 or more copies of 1q by G-banding. Locus specific FISH and spectral karyotyping were used to identify the key transient unstable and clonal structural aberrations of 1q12 resulting in segmental aneuploidies in the derivative RCs. Probe for 1q12 (Vysis) was used according to the manufacturer's protocol. Locus specific BAC clones for 1q21 (CKS1B) and 17p (TP53) were prepared and analyzed as previously described (Sawyer et al., Blood 123: 2014). IGH translocations were investigated with IGH break apart probes (Vysis). Results Data for 50 patients including CNAs of 1q21 of ≥ 4, IGH translocations, del(17p), derivative RCs, are presented. The t(4;14) was found in 15 patients, del(17p) in 23, and both aberrations were found in 8 patients. All patients showed unbalanced gains of 1q and deletions of RCs, the most frequent being 7 patients with der(1;16) and 6 with iso1q. In four of the 23 patients with del(17p), the deletion was due to a JT1q12 to 17p. Seven patients with 1q21 CNAs of ≥ 5 showed profound instability involving the 1q12 satellite DNA, demonstrating both transient and clonal aberrations driving the 1q21 CNAs. These aberrations included unstable 1q21 triplications, JT1q12s, iso1q formation with intra-arm 1q12 CNAs, and region specific breakage-fusion-bridge cycle amplifications. Conclusions Among patients with ≥ 5 CNAs of 1q21, a subset develop an acquired HR chromosome instability phenotype with an elevated rate of 1q12 pericentromeric instability characterized by concomitant deletions in 16q, iso1q, del(17p), and intra-arm segmental instability. These patients show pronounced instability in the 1q12 satellite DNA, morphologically identical to ICF syndrome, suggesting hypomethylation of this region as a driver of both 1q21 CNAs and deletions in RCs. We hypothesize that region specific hypomethylation of 1q12 provides the genomic background for the onset of an acquired 1q12 chromosome instability phenotype in MM similar to that found in ICF syndrome. For myeloma patients demonstrating this 1q12 chromosome instability phenotype we propose the term "jumping 1q syndrome." Disclosures Epstein: University of Arkansas for Medical Sciences: Employment. Davies:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; Abbvie: Consultancy; TRM Oncology: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; Janssen: Consultancy, Honoraria. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria.

Description: Although widespread skeletal dissemination is a critical step in the progression of myeloma, little is known regarding mechanisms that control metastasis of this cancer. Heparanase-1 (heparanase), an enzyme that cleaves heparan sulfate chains, is expressed at high levels in some patients with myeloma and promotes metastasis of some tumor types (eg, breast, lymphoma). Using a severe combined immunodeficient (SCID) mouse model, we demonstrate that enhanced expression of heparanase by myeloma cells dramatically up-regulates their spontaneous metastasis to bone. This occurs from primary tumors growing subcutaneously and also from primary tumors established in bone. Interestingly, tumors formed by subcutaneous injection of cells metastasize not only to bone, but also to other sites including spleen, liver, and lung. In contrast, tumors formed by injection of cells directly into bone exhibit a restricted pattern of metastasis that includes dissemination of tumor to other bones but not to extramedullary sites. In addition, expression of heparanase by myeloma cells (1) accelerates the initial growth of the primary tumor, (2) increases whole-body tumor burden as compared with controls, and (3) enhances both the number and size of microvessels within the primary tumor. These studies describe a novel experimental animal model for examining the spontaneous metastasis of bone-homing tumors and indicate that heparanase is a critical determinant of myeloma dissemination and growth in vivo.

Description: Patients with myeloma, treated on the thalidomide arm of total therapy 2 (TT2), had a higher complete response (CR) rate and improved event-free survival (EFS) but not overall survival (OS). To evaluate the benefit of TT2's posttandem autotransplant consolidation chemotherapy and dexamethasone maintenance, outcomes were compared on TT2 without thalidomide (n = 345; median follow-up, 3.5 years) and on predecessor trial TT1 (n = 231; median follow-up, 11.5 years). CR rates were similar (43% vs 41%); however, 5-year estimates of continuous CR (45% vs 32%, P 〈 .001) and 5-year EFS (43% vs 28%, P 〈 .001) were superior with TT2, with a trend for improved OS (62% vs 57%; P = .11). OS was also superior among patients achieving CR and receiving the second transplantation early after the first transplantation. Superior EFS and OS with TT2 versus TT1 was noted in the two thirds presenting without cytogenetic abnormalities (CAs); 4-year posttandem transplantation OS for patients with CAs was 47% with TT1 and 76% with TT2 when combination chemotherapy rather than DEX was applied for consolidation (P = .040). Thus, TT2 (without thalidomide) improved OS of patients without CAs; those with CAs benefited from posttransplantation consolidation chemotherapy. The favorable effects of CR and rapidly sequenced second transplantation attest to the validity of a melphalan dose-response effect in myeloma.