ALBERT

Description: Immunoglobulins (Ig) are produced by B lymphocytes as secreted antibodies or as part of the B-cell receptor. There is tremendous diversity of potential Ig transcripts (〉1 × 1012) as a result of hundreds of germ-line gene segments, random nucleotide incorporation during joining of gene segments into a complete transcript, and the process of somatic hypermutation at individual nucleotides. This recombination and mutation process takes place in the maturing B cell and is responsible for the diversity of potential epitope recognition. Cancers arising from mature B cells are characterized by clonal production of Ig heavy (IGH@) and light chain transcripts, although whether the sequence has undergone somatic hypermutation is dependent on the maturation stage at which the neoplastic clone arose. Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults and arises from a mature B cell with either mutated or unmutated IGH@ transcripts, the latter having worse prognosis and the assessment of which is routinely performed in the clinic. Currently, IGHV mutation status is assessed by Sanger sequencing and comparing the transcript to known germ-line genes. In this paper, we demonstrate that complete IGH@V-D-J sequences can be computed from unselected RNA-seq reads with results equal or superior to the clinical procedure: in the only discordant case, the clinical transcript was out-of-frame. Therefore, a single RNA-seq assay can simultaneously yield gene expression profile, SNP and mutation information, as well as IGHV mutation status, and may one day be performed as a general test to capture multidimensional clinically relevant data in CLL.

Description: Activating mutations in BRAF are found in 50% of melanomas and although treatment with BRAF inhibitors (BRAFi) is effective, resistance often develops. We now show that recently discovered NRAS isoform 2 is up-regulated in the setting of BRAF inhibitor resistance in melanoma, in both cell lines and patient tumor tissues. When isoform 2 was overexpressed in BRAF mutant melanoma cell lines, melanoma cell proliferation and in vivo tumor growth were significantly increased in the presence of BRAFi treatment. shRNA-mediated knockdown of isoform 2 in BRAFi resistant cells restored sensitivity to BRAFi compared with controls. Signaling analysis indicated decreased mitogen-activated protein kinase (MAPK) pathway signaling and increased phosphoinositol-3-kinase (PI3K) pathway signaling in isoform 2 overexpressing cells compared with isoform 1 overexpressing cells. Immunoprecipitation of isoform 2 validated a binding affinity of this isoform to both PI3K and BRAF/RAF1. The addition of an AKT inhibitor to BRAFi treatment resulted in a partial restoration of BRAFi sensitivity in cells expressing high levels of isoform 2. NRAS isoform 2 may contribute to resistance to BRAFi by facilitating PI3K pathway activation.

Description: Background: Mutations of MAP2K1, which encodes MEK1, have been identified in up to half of patients with variant Hairy Cell Leukemia (vHCL).[Waterfall et al., Nat Gen 2014, Mason et al., Leukemia & Lymphoma 2016], and have been associated with vHCL with IGHV4-34 gene usage, which This form of HCL tends to have a worse prognosis than classic HCL or wild type vHCL (Arons et al., Blood 2009), with inferior responses to chemotherapy and shorter durations of remission. Trametinib, an oral inhibitor of MEK1 and MEK2, is FDA approved for treatment of patients with BRAF p.V600E mutant melanoma. We hypothesized that this MEK inhibitor would have activity in MAP2K1 mutant vHCL. Case Report: The patient is a 52 year old man with a history of CD25+, BRAF wildtype, IGHV4-34 usage vHCL diagnosed in 2005. His previous treatments included cladribine, BL22, pentostatin/rituximab, splenectomy, single agent rituximab, ibrutinib, bendamustine/rituximab, and allogeneic transplantation from a matched unrelated donor. The patient experienced disease relapse day +350 post transplant when he developed skin nodules as well as a generalized skin rash. The skin rash appeared clinically consistent with acute GVHD. However, when biopsies of both the skin nodules and skin rash were performed he was found to have relapsed vHCL. He was consented for paired tumor and germline next generation sequencing with a 25-gene amplicon panel which revealed a somatic MAP2K1 K57N mutation that has been shown to constitutively activate MEK [Marks et al., Cancer Res 2008]. As the patient had exhausted the majority of available treatment options, he was prescribed trametinib 2 mg po daily (commercial supply, according to approved melanoma dosing). Within a week of therapy initiation his skin nodules were markedly diminished in size and his generalized rash had resolved. He did develop a new acneiform rash over his face consistent with drug toxicity. This was managed with topical agents with improvement and did not require a dose reduction. Disease restaging following cycle 2 of therapy showed near complete resolution of skin nodules, with disappearance of visible skin rash. Repeat bone marrow biopsy showed unchanged hairy cell index. Skin biopsies were repeated and phospho-ERK (T202/Y204) staining of skin biopsies pre- and post-trametinib were performed (Figure 1). This showed diminished lymphocyte involvement on H&E staining with a decrease in p-ERK expression on immunostaining, indicative of decreased signaling downstream of MEK and consistent with on target trametinib effects. As of this writing, the patient has remained on trametinib for 12 weeks with no recurrence of leukemia cutis rash. Discussion: MEK inhibition with the oral MEKi trametinib is a well tolerated therapy with clinical activity in MAP2K1 mutant vHCL. Additional studies of this agent are warranted. Optimal dose and duration of therapy will need to be explored in prospective clinical trials. Figure 1 Skin biopsies pre- and post-trametinib. (A)(C) H&E staining shows diminished lymphocyte involvement. (B)(D) PhosphoERK immunostaining shows decrease of phosphoERK expression. Bar = 500 μm Figure 1. Skin biopsies pre- and post-trametinib. (A)(C) H&E staining shows diminished lymphocyte involvement. (B)(D) PhosphoERK immunostaining shows decrease of phosphoERK expression. Bar = 500 μm Disclosures Andritsos: Hairy Cell Leukemia Foundation: Research Funding. Anghelina:Hairy Cell Leukemia Foundation: Research Funding. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding. Jones:Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Patients with Chronic Lymphocytic Leukemia (CLL) have a variety of chromosomal abnormalities and mutations. At diagnosis, about 10% of CLL patients have deletions of chromosome 17 (Del17p) leading to the loss of one allele of tumor suppressor protein TP53, which increases to over 30% in relapsed/refractory disease. Additionally, 83% of patients with a Del17p acquire a mutation on their second TP53 allele at one of several sites within the DNA binding domain. While the consequence of some of these "hotspot" mutations (R175H, R179H, G245D, G248Q/W, Y220, R213X, R273H and R282H) has been described in solid tumors and AML, very little is known of their role in CLL. Clinically, patients with Del17p/Mutp53 have worse overall survival, increased disease progression and are more likely to relapse on the current targeted therapies such as ibrutinib. Although relapse to these treatments is largely due to acquired mutations in Bruton's Tyrosine Kinase (BTK) or its downstream target PLCg2, we hypothesize that the biology of mutant 53 bearing CLL is a key driver of resistance and progression. Specifically, we aim to determine the molecular signature and downstream effectors that allow mutant p53 to drive the adverse biology associated with this subtype. Conversely, we hypothesize that targeting the mutant p53 pathway will lead to better outcomes and overall survival for patients bearing this adverse prognosis marker. We performed high-throughput Sequencing of DNA from 270 CLL patients with high coverage in the exonic regions of TP53 prior to ibrutinib therapy as well as during progression. At baseline, 40% of patients had mutations found in the DNA binding region with the most frequent occurring in R248Q, R175H and R273H. We then characterized each p53 mutant (n=106) functionally in terms of their ability to ability to activate p21, PUMA, and Bax which serve as cell cycle checkpoint and apoptotic effectors of wild type p53 in response to DNA damage. Most mutants were incompetent in upregulating p21, PUMA or Bax at the transcript level. A few mutants upregulated p21 protein in a p53 independent fashion. We then evaluated the consequence of mutant p53 in CLL. We performed chromatin immunoprecipitation (ChIP-Seq), open chromatin signatures (ATAC-Seq) and expression analysis (RNA-Seq) on CLL samples with R248Q or R175H as well as in wild-type (WT) p53 samples. Integration of ChIP, ATAC and RNA Seq profiles indicated that mutant p53 activated a unique transcriptomic profile not shared by wt p53 bearing CLL. Several genes that facilitated survival or progression were downstream targets of mutant p53. Of these, we identified PRKCB (PKC-beta), BCL2L1 (Bcl-xL), EZH2, MLL and MALAT as a potential key downstream effectors of mutant p53. To determine whether mutations at R248Q and R175H in p53 were causal in the observed increases in PKC-beta, Bcl-xL, EZH2, MLL, and MALAT we used CRISPR/Cas9 editing to introduce mutations at R175H and R248Q in the p53 wildtype CLL cell lines HG-3 and PGA-1. These were accomplished by electroporating sgRNA-Cas9 ribonucleoprotein complexes (RNPs). Western blotting of mutants revealed an increase in the mRNA and protein expression of PKC-beta, and BCL-xL in mutant p53 compared to WT. Levels of EZH2 and MLL were not increased in these cells indicating that PKC-beta and Bcl-xL may be direct transcriptional targets upregulated by mutations at R248Q and R175H in p53. Ongoing efforts will characterize the transcriptional profile of all p53 mutants in our cohort to determine whether they all have a unifying transcriptomic profile that confers a gain of function phenotype to this subtype of CLL. Disclosures Byrd: Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Acerta: Research Funding; BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau. Woyach:Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding.

Description: Introduction: Hairy Cell Leukemia (HCL) is a rare, chronic hematological malignancy that makes up approximately 2% of all leukemias. HCL patients are at a markedly increased risk for infection related to a combination of disease-related and treatment-related immunosuppression which has been well described in the literature. However, the significance of infection prior to initiation of HCL therapy and its impact on the subsequent selection of HCL treatment, or outcomes, is not well described. Using the HCL patient data registry, we report here the impact of antecedent infection on the treatment patterns and outcomes of HCL patients. Methods: We evaluated adult (≥18 years) patients with HCL who had information regarding antecedent infections and subsequent HCL treatment during 1984-2018. The primary endpoint was progression-free survival (PFS-1). Secondary endpoint included time to next treatment (TTNT). PFS-1 was measured from the date of first HCL treatment to date of progression/death or last follow-up. TTNT was defined as the time from first HCL treatment to initiation of second HCL treatment. The study population was stratified into 3 groups based on the presence or absence of antecedent infections: no infection prior to first HCL treatment (no infection group), infection within 30 days prior to first HCL treatment (infection1 group) and infection 〉30 days prior to first HCL treatment (infection2 group). Fisher's exact test or Kruskal-Wallis test was used to compare the characteristics among the no infection and infection groups and the Cox proportional hazard model was used to evaluate the association with PFS-1 and TTNT. Results: A total of 205 HCL patients who had information regarding antecedent infections and subsequent HCL treatment were eligible for the study. Among these, 144 (70%) belonged to the no infection group, while 26 patients (13%) belonged to infection1 group and 35 (17%) to infection2 group. Patient characteristics are shown in Table 1 with a breakdown between the three groups. The majority of the patients were Caucasian with a male preponderance and had classic HCL. The patients in the infection1 group had a lower median WBC (K/uL) (1.9 vs 3.1 vs 2.9), particularly the absolute neutrophil count (K/uL) (0.4 vs 0.7 vs 0.8) and significantly lower median hemoglobin (gm%) (10.1 vs 12.2 vs 12.4) relative to the no infection and infection2 groups, respectively (p=0.01). Similarly, a greater proportion of patients in the infection1 group had significant comorbidities (including pulmonary, gastrointestinal and hepatic disease) relative to no infection and infection2 groups as shown in Table 1. The majority of patients received purine nucleoside analogs as their first HCL treatment (no infection group=92%, infection1 group=85%, infection2 group=94%). The median PFS-1 (in years) was better in the no infection group compared to the infection1 group but was not statistically significant (17.0 [95% CI=7.9-not reached (NR)] vs 8.8 [95% CI=4.2-NR], respectively, p=0.98, Figure 1). However, the median TTNT (in years) was significantly longer for HCL patients with no infection versus the infection1 group (6.3 [95% CI=5.4-7.8] vs 3.6 [95% CI=0.7-NR], respectively, p=0.001, Figure 1). On subgroup analysis, relative to the no infection group, median PFS-1 (in years) was not significantly different in infection1 group treated with Pentostatin (10.7 [95% CI=3.53-NR] vs NR [95% CI=1.38-NR], respectively, p=0.43), however, the median PFS-1 (in years) was shorter in the infection1 group treated with Cladribine (17.0 [95% CI=7.67-NR] vs 4.0 [95% CI=2.00-NR], respectively), although not reaching statistical significance (p=0.09) probably due to small sample size. Conclusion: In this large series of HCL patients who received treatment, we show that the patients who had infections at the time of HCL treatment have a significantly shorter TTNT. The reasons for this are unclear but may indicate that patients were unable to receive treatment in a timely manner because of the infection, or were unable to complete treatment because of complications. The significant difference in hemoglobin between the infection1 and other groups indicates the possibility that these patients had more advanced HCL at the time of diagnosis. These findings indicate the potential long term negative impact of infections in patients who need treatment for HCL and reinforce the need for careful management in this setting. Disclosures Lozanski: Beckman: Research Funding; Coulter: Research Funding; Stem Line: Research Funding; Genentech: Research Funding; Novartis: Research Funding; BI: Research Funding. Andritsos:HCLF: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy.

Description: Alterations in global DNA methylation patterns are a major hallmark of cancer and represent attractive biomarkers for personalized risk stratification. Chronic lymphocytic leukemia (CLL) risk stratification studies typically focus on time to first treatment (TTFT), time to progression (TTP) after treatment, and overall survival (OS). Whereas TTFT risk stratification remains similar over time, TTP and OS have changed dramatically with the introduction of targeted therapies, such as the Bruton tyrosine kinase inhibitor ibrutinib. We have shown that genome-wide DNA methylation patterns in CLL are strongly associated with phenotypic differentiation and patient outcomes. Here, we developed a novel assay, termed methylation-iPLEX (Me-iPLEX), for high-throughput quantification of targeted panels of single cytosine guanine dinucleotides from multiple independent loci. Me-iPLEX was used to classify CLL samples into 1 of 3 known epigenetic subtypes (epitypes). We examined the impact of epitype in 1286 CLL patients from 4 independent cohorts representing a comprehensive view of CLL disease course and therapies. We found that epitype significantly predicted TTFT and OS among newly diagnosed CLL patients. Additionally, epitype predicted TTP and OS with 2 common CLL therapies: chemoimmunotherapy and ibrutinib. Epitype retained significance after stratifying by biologically related biomarkers, immunoglobulin heavy chain mutational status, and ZAP70 expression, as well as other common prognostic markers. Furthermore, among several biological traits enriched between epitypes, we found highly biased immunogenetic features, including IGLV3-21 usage in the poorly characterized intermediate-programmed CLL epitype. In summary, Me-iPLEX is an elegant method to assess epigenetic signatures, including robust classification of CLL epitypes that independently stratify patient risk at diagnosis and time of treatment.

Description: Key PointsAEB071 demonstrates preclinical in vitro and in vivo activity against CLL independent of survival signaling and stromal cell protection. AEB071 can either inhibit or activate the WNT pathway emphasizing the importance of pharmacodynamic monitoring in its development.

Description: Introduction: Exportin-1 (XPO1), a nuclear transport protein critical for the export of tumor suppressor proteins (TSPs) and select mRNAs to the cytoplasm, is highly expressed in acute myeloid leukemia (AML) and correlates with poor survival. Selinexor, an oral, first-in-class, selective inhibitor of nuclear export, blocks XPO1 function. We previously reported that sequential treatment of AML blasts using the hypomethylating agent decitabine followed by selinexor exhibited strong anti-leukemic effects in vivo by inducing the expression of silenced TSPs that are kept in the nucleus by XPO1 inhibition (Ranganathan, Blood 2015). Methods: Based on these findings, a phase I dose-escalation study was initiated to evaluate the safety, feasibility, maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), and preliminary clinical activity of selinexor in combination with decitabine in poor-risk AML pts (NCT02093403). Adults with relapsed or refractory (R/R) AML and older (age ≥60) unfit pts with untreated AML were eligible. Pts received 10-day decitabine induction(s) at 20mg/m2 on days 1-10 for up to four 28-day cycles in combination with selinexor once daily, twice weekly beginning on day 11. Pts with

Description: Background:Spleen tyrosine kinase (SYK) is a nonreceptor cytoplasmic tyrosine kinase primarily expressed in cells of hematopoietic lineage. Constitutive activation of SYK in acute myeloid leukemia (AML) has been reported and targeted inhibition of SYK induced differentiation in vitro and demonstrated anti-leukemia activity in AML mouse models. SYK has also been shown to directly phosphorylate the FLT3 receptor, modulating its activation and possibly promoting its role in leukemogenesis. Entospletinib is an orally bioavailable, selective inhibitor of SYK shown to be clinically active in B-cell malignancies. Here we evaluate the combination of entospletinib in patients with untreated AML using a 14-day window phase to assess single-agent activity, then adding standard intensive chemotherapy. Methods: In this phase 1b/2 study (NCT02343939), patients age 18 to 70 years with previously untreated AML, preserved organ function, and ECOG ≤ 2 were eligible to receive dose escalated entospletinib for 14 days as monotherapy (days -14 to 0) followed by combination with daunorubicin 60 mg/m2/d, cycle 1 day 1 to 3, and cytarabine 100 mg/m2/d, cycle 1 day 1 to 7. All patients received entospletinib monotherapy for up to 14 days prior to starting induction. Chemotherapy could be initiated after 5 days of monotherapy (and entospletinib continued for 4+ weeks) in patients with leukemia-related complications necessitating chemotherapy. Patients enrolled to dose level (DL) 0 and DL 1 received entospletinib 200 mg po BID and 400 mg po BID, respectively. Patients with residual disease two weeks after chemotherapy received a second induction cycle identical to the first. Entospletinib was continued without interruption until remission was assessed at count recovery. Results:Twelve patients enrolled with a median age of 54 (range, 18-69) years. Patients were in the following European LeukemiaNet genetic risk groups: favorable (n=1), intermediate I (n=3), intermediate II (n=2), and adverse (n=4), respectively. Three patients were not evaluable for dose limiting toxicity (DLT) assessment and were replaced (due to detection of CNS disease requiring non-study therapy (n=1), and withdrawal of consent unrelated to drug toxicity (n=2)). Single-agent entospletinib during the window period was well tolerated; toxicities after combination with intensive chemotherapy were common and typical. Among 3 patients treated at 200mg BID, no DLT was observed. Of 3 patients treated at 400mg BID, a patient with documented fungal pneumonia developed grade 3 pneumonitis that was possibly related to entospletinib. Although this did not meet DLT criteria, DL 1 was expanded with 3 additional patients, none of whom experienced DLT. Overall, the most common non hematologic adverse events (inclusive of intensive chemotherapy periods) were febrile neutropenia, nausea, and diarrhea. Based on this clinical experience and compiled pharmacokinetic data demonstrating lack of benefit to further dose escalation, 400 mg BID was selected as the recommended phase 2 dose. Responses were seen at both levels. Among the 3 patients treated at 200 mg BID, two required a second induction but all achieved a complete remission (CR) (3/3; 100%). Of the 6 patients treated at 400mg BID, none required a second induction and the CR rate was also 100%. Remarkably, an 18 year old male with 11q23-rearranged AML achieved morphologic and cytogenetic CR after only the 14 day entospletinib monotherapy window (prior to chemotherapy). Another patient with 11q23-rearranged AML had significant platelet response during the window period (this patient refused disease evaluation by marrow aspiration prior to chemotherapy). Conclusions: Entospletinib appears to have significant clinical activity in AML, and its combination at doses up to 400mg BID with intensive chemotherapy is well tolerated. An extended phase 2 program is now underway. Patients with 11q23-rearranged AML may be uniquely sensitive to SYK inhibition by entospletinib. Detailed molecular analysis of these patients is ongoing and will be presented. Disclosures Walker: Gilead Sciences: Research Funding. Bhatnagar:Karyopharm: Research Funding. Marcondes:Gilead Sciences: Employment, Equity Ownership. DiPaolo:Gilead Sciences: Employment, Equity Ownership. Abella-Dominicis:Gilead Sciences: Employment, Equity Ownership.