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PurposeThis work tries to shed light on what factors can influence, positively or negatively, the decision to license a patent from a university, in order to offer some recommendations that can contribute to increasing the number of patents licensed from universities.Design/methodology/approachBased on a sample of researchers at Spanish universities who have already registered patents, this work shows that the individual factors of the researcher outweigh the institutional factors in determining the decision to patent an invention. Likewise, the probability of patenting an invention is higher when the researcher's level of participation in the process is greater.FindingsThe results of our study allow us to affirm that, in the Spanish university setting, individual factors play a more important role in one's decision to license a patent than institutional factors. In this sense, the collaboration of companies or experts from outside of academia in the research from which the patent was granted is the most relevant factor.Originality/valueThis work, the first study of this type to be carried out in Europe, concludes with a recommendation for reinforcing the structure and functionality of technology transfer offices as a basic policy for the promotion and facilitation of commercial exploitation of innovation in the universities.
Abstract In this article, we investigate whether academic technology-based knowledge crosses university boundaries or remains trapped inside the ivory tower. To do so, we rely on a matched sample approach to compare the spillovers generated by academic and firm patents using measures that take into account knowledge spilling-in and knowledge spilling-out of academia. Although it is true that knowledge exchanges among universities may inflate the overall spillovers of university patents vis-à-vis firm patents, our results indicate that university patents generate more spillovers than a comparable sample of matched corporate patents, even when knowledge flows among universities are not regarded as spillovers. This suggests that, in our sample, firm technologies more frequently rely on academic patents than on technologies from other corporations. In addition, we find that the gap between university and industry spillovers differs across industries, with industries where patents are important for appropriating returns from R&D (i.e. more economically valuable), such as drugs, presenting a smaller gap than in industries with complex technologies (where firms have strong incentives to patent aggressively), such as computers. Finally, we show that industry patents generate more spillovers locally and that academic knowledge spillovers are less geographically localized than those of corporate research.
Especialización vs diversificación del conocimiento tecnológico universitario en las regiones europeas
This paper explores the relationship between regional patterns of university technological diversification/specialization in Europe and its effects on the production of new university technological outcomes as measured by patent counts. Our dataset contains 4,580 university-owned patents related to 202 regions of European Union-15 for the period from 1998 to 2004. Several econometric specifications reveal that a diversification strategy in the production of university technological knowledge favours the generation of subsequent new university patents. Specialization has a positive and significant effect in high-technology sectors.
Introduction: While treatments for CLL have improved in recent years, CLL remains incurable for most patients who often rely on long-term suppressive medications. These can present complications associated with undesirable side effects and the risk of relapse. NK cell therapy holds great promise due to NK cells' powerful innate anti-tumor effects, with the potential to induce deep remission or even cure. However, previous efforts have been constrained by low cell numbers and limited cytotoxicity against CLL cells. Stimulating NK cells ex vivo with K562-based feeder cells expressing membrane-bound IL-21 (mbIL-21) induces high levels of expansion and activation, with potent cytotoxicity against various tumor cells (Denman et al. PLoS ONE 2012). We have recently demonstrated that allogeneic NKs from normal donors, expanded using mbIL-21, are potently cytotoxic to CLL cells (Yano et al. iwCLL 2019). Here, we test this technique with autologous NK cells. Autologous therapy will allow the benefits of administering activated NK cells without the risks of immunosuppression required for allogeneic treatment. Methods: We isolated NK cells from CLL patient blood and expanded them for 21 days using IL-21 expressing feeder cells and IL-2 (Denman et al. PLoS ONE 2012). We then characterized the cytotoxic capacity of the CLL-derived expanded NKs (CLL-XNKs) using calcein release assays. We measured both direct cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) against OSU-CLL and Mec1 CLL cell lines, allogeneic primary CLL cells, and autologous CLL cells. We compared CLL-XNK cells to unstimulated NK cells from normal donors and to normal donor-derived expanded NKs (ND-XNKs), produced using the same protocol. Results: During mbIL-21 NK stimulation, CLL-derived NK cells underwent an average of 5,900-fold expansion and maintained exponential growth throughout the 21-day expansion. This growth is similar to normal donor-derived NK cells (doubling time 1.6 days for CLL-derived vs. 1.5 days for donor-derived, p = 0.26, n=5). Cytotoxicity data comparing CLL-XNK cells versus ND-XNKs and unstimulated donor-derived NK cells is included in Table 1. We tested a range of effector:target ratios and found a dose response pattern of increasing cytotoxicity from 0.3125:1 to 10:1 ratios. Interestingly, while ADCC with either antibody was superior to direct cytotoxicity, obinutuzumab was not superior to rituximab for stimulating CLL-XNK cytotoxicity, differing from our experiences with ND-XNKs (Yano et al. iwCLL 2019). First, we demonstrate that CLL-XNK cells show potent cytotoxicity against both OSU-CLL and Mec1 CLL cell lines, via both direct cytotoxicity and ADCC (Table 1). These results show CLL-XNK cells to be similar or greater in potency in comparison to both ND-XNKs and normal unstimulated NKs. CLL-XNK cells also show cytotoxicity against allogeneic primary CLL cells, with greater cytotoxic activity than unstimulated, normal donor-derived NK cells (Table 1). Interestingly, while ADCC with obinutuzumab was similar between CLL-XNKs and ND-XNKs (p=.44), direct cytotoxicity and rituximab-induced cytotoxicity were both higher with CLL-XNKs than ND-XNKs (p=.0001 and .041) (Table 1). These results contrast with previous reports that LAKs derived from CLL patients have decreased potency (Foa et al. and Santiago-Schwarz et al. Blood 1990). Finally, CLL-XNKs showed potent cytotoxicity against autologous CLL cells using both direct cytotoxicity and ADCC (Table 1). Conclusion: We have successfully expanded NK cells from CLL patients and demonstrated their cytotoxicity against CLL cell lines, unmatched CLL cells, and autologous CLL cells. These patient-derived cells are superior to normal unstimulated NKs and are similar or even better than expanded donor NK cells. Ongoing studies will explore in vivo function of these NK cells, combination with CLL-targeted treatments, and further functional measures. IL-21-expanded NK cells represent a promising new therapy for CLL in both allogeneic and autologous settings. (*MY and JRL contributed equally to this work. MY is a recipient of a Pelotonia Graduate Fellowship and JRL is a recipient of a Hendrix Summer Scholars Fellowship. This work was supported by NIH R35 CA197734.) Disclosures Lee: Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Muthusamy:Ohio State University: Patents & Royalties: OSU-2S. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau.
Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by genetic dysregulation of cyclin D1 and activation of signaling pathways driving uncontrolled MCL cell proliferation and survival. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK), a downstream target of the B-cell receptor (BCR) pathway. While ibrutinib exhibits significant single-agent therapeutic activity in patients with relapsed/refractory MCL, the vast majority of MCL patients on ibrutinib progress with aggressive disease and short survival (3-8 mo). Although ~80% of chronic lymphocytic leukemia patients with acquired ibrutinib resistance have mutations in BTK and PLCγ2, this is uncommon in MCL suggesting alternative mechanisms driving this resistant phenotype. Understanding drug-resistance mechanisms and developing effective therapies for ibrutinib resistant (IR) MCL are urgently needed. The major type II protein arginine methyltransferase enzyme, PRMT5, catalyzes symmetric dimethylation of arginine residues on histone tails (H3R8 and H4R3) and other proteins. PRMT5 regulates a vast array of biologic functions including RNA processing, DNA damage response, signal transduction, and gene expression. Amplified PRMT5 activity drives the expression and activity of key oncogenes (MYC, CYCLIND1, NOTCH1) while silencing expression and activity of tumor suppressors (ST7, RBL2, and p53). Our group has shown PRMT5 is overexpressed and dysregulated in MCL and strategies aimed at selectively targeting PRMT5 show anti-tumor activity in preclinical lymphoma models. Here we describe the development of a novel patient derived xenograft (PDX) of IR-MCL and explore PRMT5 inhibition as an alternative therapeutic option to circumvent IR. Peripheral blood mononuclear cells from a 75 yo male patient diagnosed with acquired classic IR-MCL were engrafted intravenously into NSG mice. After 5 passages, all mice engrafted with 107 MCL cells developed histologically confirmed MCL infiltrating kidney, lymph nodes, bone marrow, spleen and peripheral blood. Circulating human CD5+/CD19+ cells were detectable and quantifiable by flow cytometry by day 21 post-engraftment. Karyotype analysis confirmed the hallmark t(11;14)(q13;q32) of MCL while retaining nearly all cytogenetic abnormalities present in the patient's primary tumor including a deletion of chromosome 9, associated with deletion of MTAP, a therapeutic vulnerability for PRMT5-targeted therapy. Whole exome sequencing confirmed genomic stability with successive passages. Ex vivo cytotoxicity assays and protein pathway analysis further confirmed resistance to ibrutinib (IC50 >1 µM) with maintained hyper-phosphorylation of AKT (Ser473) and ERK (Thr202/Tyr204). Western blot analysis showed elevated levels of c-MYC, CYCLIND1, BCL2, and pERK. After validation of circulating disease at day 25 post engraftment, mice were treated with either a novel small molecule inhibitor of PRMT5 (PRT382, 10 mg/kg orally 4 days on 3 days off) or ibrutinib (75 mg/kg administered in drinking water, n=5 mice per treatment group). Treatment of this PDX model with PRT382 resulted in significantly decreased disease burden and improved median survival compared to control animals from 48 to 83 days, respectively (p=0.0045). We found no significant difference in survival (p= 0.6540) or circulating disease burden with ibrutinib therapy compared to control group. The full BTK occupancy of ibrutinib treated mice was validated using fluorescence resonance energy transfer-based assay. Ex vivo PDX MCL cells from PRT382-treated mice showed loss of symmetric dimethyl arginine with preservation of asymmetric dimethyl arginine levels, reduced H4(Sme2)R3 epigenetic marks, and elevated levels of BCL2, MYC, and pAKT/pERK. We developed a cell line (SEFA) allowing for in vitro mechanistic studies. We are currently investigating potential mechanisms responsible for circumventing IR-MCL by integrating genome-wide changes to chromatin accessibility and whole transcriptome analysis. This IR-MCL PDX mouse model serves as a useful tool to investigate mechanisms of drug resistance, provides a platform to explore novel pre-clinical therapeutic strategies to circumvent IR and demonstrates the therapeutic activity of PRMT5 targeted therapy in this aggressive disease. Disclosures Byrd: Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.
Introduction: Targeted irreversible Bruton's Tyrosine Kinase (BTK) inhibitors ibrutinib and acalabrutinib, have revolutionized treatment for chronic lymphocytic leukemia (CLL). While BTK inhibition (BTKi) achieves durable responses in 90% of patients, only 10% achieve minimal residual disease (MRD) negative status. MRD positive patients have persistent residual CD5+CD19+ tumor B cells at approximately 1-5 /mm3 in peripheral blood. These cells may represent a subpopulation of B-cell lymphocytosis pre-malignant cells or may carry a BTK C481, PLCG2, or other CLL mutation that is ultimately responsible for disease relapse. Alternatively, MRD could be derived from the original clones present at initial disease presentation that are not dependent on BTK signaling. Readily available clinical DNA sequencing and MRD monitoring techniques lack the ability to characterize these cells adequately due to their rarity in peripheral blood. To address this problem, we developed a novel method for limited-cells using fluorescence activated cell sorting in tandem with next generation sequencing (LC-FACSeq) to characterize rare tumor subpopulations in the blood and bone marrow. LC-FACSeq may be useful not only for CLL but also other leukemias. Methods: LC-FACSeq uses fluorescent activated cell sorting (FACS) to isolate pure populations of rare tumor cells after which targeted deep sequencing is performed to monitor CLL-related mutations in NOTCH1, SF3B1, and TP53, as well as genes associated with BTKi relapse and resistance: BTK and PLCG2. For validation of this method, we generated libraries from DNA isolated from FACS isolated bulk (n >15000) versus n= 50, 100, 300, or 500 CD5+/CD19+ cells from CLL patients (n=5). Results: All samples analyzed had an average read depth of 1212 (SEM=56) per gene and an average coverage uniformity of 88.24% (SEM=.01). We show that showed that 300-cell LC-FACSeq libraries demonstrated comparable variant calling and minimal noise to standard libraries generated from purified DNA from bulk cells. Using samples from patients with previously identified BTK C481S mutations, we found that both sensitivity and specificity of LC-FACSeq for BTK C481S was 100%. Furthermore, LC-FACSeq reliably amplified BTK C481S signals from subclones as small as 6 in 300 total cells (2%) when mutated tumor cells were serially diluted into BTK wild type tumor cells. In using LC-FACSeq to retrospectively analyze four independent patients who developed Ibrutinib resistance, we found that we could see the emergence of small BTKi resistant subclones as early as 10 months before clinical detection. We next extended LC-FACSeq to examine the clonal architecture of long-term (> 12 months) ibrutinib-treated MRD positive patients. Median treatment time was 5 years. BTK C481S mutations were observed in the latest available on-treatment samples of only one patient. Using LC-FACSeq we observed canonical CLL-associated clonal mutations similar to those observed in previous studies. Of the 14 MRD positive patients, 7 showed subclonal changes in TP53, NOTCH1, POT1, SF3B1, and MYD88 over the course of ibrutinib treatment although we found no correlation or consensus in these clonal shifts. Conclusion: LC-FACSeq is a highly sensitive method of characterizing clonal evolution in rare cells. Our data shows that LC-FACSeq is useful for monitoring sequential acquisition of mutations conferring therapy resistance and clonal evolution in long-term ibrutinib treated chronic lymphocytic leukemia (CLL) patients. We also observe that in most cases, MRD clones after long-term ibrutinib treatment are genetically similar to disease clones from pretreatment baseline. Compared to current MRD monitoring strategies, the main advantages of LC-FACSeq are that 1) variants can be confidently called from rare sorted tumor populations and subpopulations, 2) library generation can be completed in less than a day in a diagnostic laboratory compared to the labor-intensive protocols of traditional NGS approaches, and 3) amplicon panels can be easily customized for application to other types of leukemia and lymphoma. (EH is supported by the Graduate Pelotonia Fellowship and the NIH F30) Disclosures Bhat: Janssen: Consultancy; Pharmacyclics: Consultancy. Rogers:Janssen: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Acerta Pharma: Consultancy. Woyach:AbbVie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding. Lozanski:Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentec: Research Funding; Boehringer Ingelheim: Research Funding. Muthusamy:Ohio State University: Patents & Royalties: OSU-2S. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Genentech: Research Funding; Acerta: Research Funding.
Introduction: Treatment of chronic lymphocytic leukemia (CLL) has been transformed by small molecule inhibitors targeting the B-cell receptor (BCR) signaling cascade. The first-in-class small molecule inhibitor of Bruton's Tyrosine Kinase (BTK), ibrutinib, is FDA approved as a frontline therapy for CLL. However, resistance to BTK inhibition has emerged in patients through acquisition of mutations in BTK or its immediate downstream target, PLCG2, emphasizing the need for alternative targets and therapies. BCR signaling remains intact in the presence of these mutations, making targeted inhibition of proteins downstream of BTK an attractive therapeutic strategy. Protein kinase C-β (PKCβ) is a downstream member of the BCR signaling pathway that we have previously demonstrated as an effective therapeutic target in CLL. MS-553 is a potent, ATP-competitive, reversible inhibitor of several PKC isoforms including PKCβ. Therefore, we evaluated the effects of MS-553 in primary CLL cells. Methods: Primary CLL cells were isolated by negative selection and treated with increasing concentrations of MS-553 to a maximum dose of 10 µM. BCR signaling changes were interrogated by change in target protein phosphorylation by immunoblot following a 24 hour drug incubation with and without phorbol ester stimulation (90 minutes) in CLL samples. Inhibition of CpG-mediated activation of CLL cells was measured using flow cytometry (CD86 and HLA-DR) in ibrutinib refractory patient samples at baseline and post-relapse due to the emergence of the p.C481S BTK mutation. CCL3 and CCL4 expression was measured by ELISA after 24 hours in primary CLL cells in the presence or absence of anti-IgM ligation. TNFα expression was also measured by ELISA in negatively selected, healthy donor T cells treated with MS-553 for 24 hours with or without anti-CD3 and anti-CD28 stimulation. Results: At 24 hours, 5 µM MS-553 inhibited downstream BCR signaling in primary CLL cells, demonstrated by 31% reduced phosphorylation of PKCβ (p=0.08, n=5) and several of its downstream targets including GSK3β (40%, p<.01, n=5) , ERK (46%, p=0.02, n=4) , and IκBα (56%, p=0.04, n=5) compared to vehicle treated, stimulated samples. CpG-mediated TLR9 stimulation increases expression of CD86 and HLA-DR in primary CLL cells. In baseline samples from ibrutinib treated patients, 10 µM MS-553 decreased expression of CD86 by 34% and HLA-DR by 91%. In matched patient samples post-relapse due to ibrutinib resistance, MS-553 (10 µM) maintained the ability to decrease expression of CD86 (49%) and HLA-DR (84%). Pro-inflammatory cytokine expression by primary CLL cells stimulated with anti-IgM decreased in the presence of 5 µM MS-553, with CCL3 decreasing by 36% (p=0.06, n=5) and CCL4 decreasing by 79% (p<.01, n=4) compared to vehicle treated, stimulated controls. TNFα expression by healthy T cells increased with anti-CD3 and anti-CD28 stimulation; 1 µM MS-553 reduced TNFα expression by 97% compared to vehicle treated, stimulated controls (p<.01, n=9). Conclusions: MS-553 is a novel and potent inhibitor of PKC demonstrating in vitro efficacy in CLL. MS-553 is able to inhibit BCR signaling by blocking phosphorylation of PKCβ and its downstream targets. CpG-mediated activation is reduced with MS-553 treatment in ibrutinib refractory patient samples both at baseline and post-relapse. Inflammatory signaling by primary CLL cells is further abrogated by MS-553 in its ability to decrease CCL3 and CCL4 cytokine expression. In an ongoing phase I clinical trial of MS-553, patient samples show a potent and dose dependent decrease in PKCβ activity as measured by a clinical biomarker assay. Together, our results suggest that MS-553 targets PKCβ in primary CLL to inhibit signaling and survival, establishing MS-553 as a potential therapeutic for treating CLL. These data justify continued preclinical and clinical work in the development of MS-553 for the treatment of CLL. Disclosures Niesman: MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zhang:MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Byrd:BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Genentech: Research Funding; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding. Woyach:Verastem: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding.
Normal hematopoiesis is organized in a hierarchical manner and it has been hypothesized that acute myeloid leukemia (AML) is organized in a similar way with leukemia-initiating cells (LIC) at the top of the hierarchy, giving rise to more differentiated blasts to sustain AML. Therefore, elimination of LIC population is critical for cure. This may be accomplished via novel molecular targeted therapies. The mutational composition of LIC and non-LIC compartments in AML has not been fully elucidated and could provide new insights into biology and treatment. We investigated the distribution and variant allelic frequencies (VAFs) of recurrent gene mutations within these compartments in newly diagnosed CD34+ AML patients (pts). We studied a total of 88 pts. CD34- AML cases, defined as <5% positivity on blasts, were excluded. Pre-treatment bone marrow or apheresis samples were sorted and sequenced with our ultrasensitive limited cell (LC)-FACS-seq method. First, we gated on CD45dimLin- leukemic population, followed by isolation of 300 cells from CD34+CD38- (LIC), CD34+CD38+ (non-LIC) and CD34- compartments. To compare with the bulk population, DNA was extracted from 500,000 CD45dimLin- leukemic blasts. All samples were sequenced with a 27-gene targeted panel. Extreme Limited Dilution Analysis (ELDA) platform was used for colony formation assays and estimation of stem cell frequencies. Clinical characteristics are summarized in Table 1. The median frequency of the LIC population was 0.5% (range, 0.01% - 69%). The prevalence of high LIC frequency (≥0.5%) was significantly higher in pts with adverse risk (AR) AML, as compared to intermediate (IR) and favorable risk groups (94% vs 34% vs 16%, respectively, p<.001). When compared to pts with low LIC frequency (<0.5%), those with high LIC frequency had worse overall survival (median, 9 months vs not reached, p=.003) and relapse-free survival (median, 4 vs 15 months, p=.01). In 10 pts who had serial relapse samples, LIC frequencies were increased at the time of relapse (p=.03). We re-validated the commonly used LIC markers with ELDA of primary AML cells. In one IR sample, stem cell frequencies in sorted CD34+CD38-, CD34+CD38+ and CD34- compartments were 1:3, 1:15 and 1:16, respectively (p<.001). In one AR sample, stem cell frequencies were 1:1, 1:8, and 1:12, respectively (p<.001). Using these markers, LICs and non-LICs were enriched and sequenced. The average number of mutations detected by sequencing of bulk samples was significantly lower than sorted LIC (3.17 vs 3.75, p<.05) and non-LIC (3.17 vs 3.96, p<.001) populations indicating the higher sensitivity of our method in detecting subclonal mutations. Mean VAFs were similar between LIC and non-LIC populations for NPM1 (42% vs 47%), DNMT3A (37% vs 41%), IDH1 (41% vs 48%), IDH2 (43% vs 48%), and U2AF1 (37% vs 42%) mutations. Mutations involving signaling pathways were more frequent in non-LICs, including FLT3-TKD (12% vs 23%, p<.01), NRAS (17% vs 26%, p<.01) and KRAS (13% vs 19%, p<.05) mutations, which might be explained by their later acquisition during AML development. In addition, among 22 pts with CEBPA mutation, 16 (73%) harbored the mutation exclusively in non-LICs. Finally, 13 pts with TP53 mutations had different VAFs between compartments. Among 4 pts who had doubling of VAF from LIC to non-LIC compartment, 3 had subclones with del(17p) in LIC pool detected by FISH. LIC subclones harboring both del(17p) and TP53 mutation (i.e. loss of heterozygosity) propagated to drive leukemia. Relapse samples obtained from 6 pts were analyzed and compared with diagnosis. In all cases, we could identify LIC clones that persisted after chemotherapy and led to relapse (see example in Figure). Similarly, 3 pts who were primary refractory showed persistence of LIC clones that were resistant to treatment. On the contrary, 6 pts in whom LIC clones could be eradicated with treatment did not experience disease recurrence. LICs exist at a very low frequency in pre-treatment AML samples. The mutational composition of LIC-enriched compartment shows differences from blasts constituting the bulk of leukemia, which is consistent with the sequence of mutations observed during the evolution of AML. LC-FACS-seq is an ultrasensitive method to detect mutations in a tiny population of residual LICs in pts at remission. Therapies targeting mutations that are concentrated in LICs may re-shape the clonal hierarchy and impact on disease course. Disclosures Behbehani: Fluidigm corporation: Other: Travel funding. Byrd:Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentec: Research Funding.
Monoclonal B cell lymphocytosis (MBL) has been shown to be the precursor condition that precedes overt diagnosis of chronic lymphocytic leukemia (CLL). Whereas CLL is classified with greater than or equal to 5 × 109/L B lymphocytes in the peripheral blood, MBL is a clonal expansion of B-cells below this threshold. MBL can be further divided into high- or low-count based on whether the B-cell count is above or below 0.5 × 109/L. Approximately 10% of the population over 40 is estimated to have MBL, increasing to >50% over the age of 90. While low-count MBL is unlikely to progress, 1-2% of high-count (HC) MBL individuals progress to CLL requiring therapy per year. It is debatable if all patients with detectable MBL should be classified as an entity requiring monitoring by a hematologist, especially for low-count MBL. In addition, the diagnosis of leukemia is distressing to patients; therefore, it is important to identify HC MBL patients that are more likely to progress to disease requiring treatment and thus should be monitored more closely. As the majority of MBL cases phenotypically resemble CLL, established prognostic markers including recurrent chromosomal aberrations, beta-2 microglobulin levels, and the mutational status of the Immunoglobulin heavy-chain variable region locus (IGHV) have been shown to predict time to treatment (TTT) and overall survival (OS) in a large retrospective study of MBL1. CLL patients can also be divided into three distinct epigenetic subtypes that reflect progressive DNA methylation changes that occurs during B cell development. These 'epitypes' termed low-programmed (LP), intermediate-programmed (IP), and high-programmed (HP) independently predict clinical outcomes irrespective of disease stage and treatment2. LP-CLL patients follow a generally unfavorable clinical course compared to the more indolent HP-CLL patients, while IP CLL patients display an intermediate outcome. Here we sought to determine if epitype forecasts progression to CLL and eventual clinical outcome for individuals with MBL. We analyzed 66 individuals diagnosed with HC MBL at the Mayo Clinic with a median follow-up of 6.3 years. Developmental epitype was determined using our novel Methylation-iPLEX technique that interrogates 34 CpGs and assigns epitype using a random forest model2. Seventy-seven percent of the MBL cases were assigned to one of the three epitypes: 42.4% HP, 19.7% IP, and 15.2% LP. The residual 23% remained unclassified due to ambiguous (low confidence) epigenetic patterns or insufficient purity (Figure 1A). The overall proportion of HP and IP epitypes in MBL were significantly greater than proportions observed in CLL cohorts (P<0.01). Epitypes remained stable over time as 20/21 cases for which we obtained a high-confidence epitype classification at multiple time points remained unchanged. Epitype significantly predicted MBL individuals progressing to treatment (P=0.001) with LP individuals progressing in a median of 5.6 years (P=0.049 and P=0.0002 versus IP and HP, respectively); median was not reached in HP or IP (Figure 1B). Epitype also predicted overall survival (OS) in MBL (P=0.04), with LP individuals having a significantly shorter OS (median of 11 years versus not reached in IP and HP; P=0.056 and P=0.048 versus IP and HP, respectively). In this study we evaluated a cohort of 66 HC MBL cases and determined that classification using developmental DNA methylation epitypes can be employed in HC MBL to aid in risk stratification. HC MBL patients classified as LP are more likely to progress to requiring treatment and have a significantly reduced OS. The epigenetic classification may help clinicians decide how closely and frequently a HC MBL individual needs to be monitored. Figure 1: (A) Breakdown of the epigenetic subtype assigned to 66 HC MBL samples. (B) Kaplan-Meier analysis of time to treatment and (C) overall survival of MBL patients separated by epitype. 1. Parikh, S. A. et al. Outcomes of a large cohort of individuals with clinically ascertained high-count monoclonal B-cell lymphocytosis. Haematologica103, e237-e240 (2018). 2. Giacopelli, B. et al. Developmental subtypes assessed by DNA methylation-iPLEX forecast the natural history of chronic lymphocytic leukemia. Blood blood.2019000490 (2019). doi:10.1182/blood.2019000490 Disclosures Byrd: Ohio State University: Patents & Royalties: OSU-2S; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding. Shanafelt:Patent: Patents & Royalties: US14/292,075 on green tea extract epigallocatechin gallate in combination with chemotherapy for chronic lymphocytic leukemia; Merck: Research Funding; Polyphenon E International: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Glaxo-SmithKline: Research Funding; Abbvie: Research Funding; Cephalon: Research Funding; Celgene: Research Funding. Parikh:Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding. Kay:Agios: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board.
Background: Aberrant expression levels of several miRs have been reported to independently associate with outcome of patients (pts) with CN-AML. In these reports, miR expression was profiled using microarray assays, which interrogate a selected subset of miRs. The advent of next-generation sequencing (NGS) has allowed unbiased measurement of miR expression, but, to our knowledge, NGS has not been used to identify miRs associated with prognosis of AML pts. Here, we analyze small RNA sequencing (smRNA-seq) data from a large cohort of younger adults with CN-AML, for whom outcome data were available, with the goal to identify new prognostic miRs. Methods: We performed smRNA-seq in 281 younger adults (aged 18-59 y) with de novo CN-AML. Cytogenetic analyses were performed in Cancer and Leukemia Group B (CALGB)/Alliance institutional laboratories using standard banding techniques; mutational analyses were done centrally using a targeted DNA sequencing platform. All pts were treated on frontline CALGB/Alliance protocols. Results: We first evaluated which miRs associated with overall survival (OS) in univariable analysis; we detected 9 such miRs. We then used a machine-learning approach, namely random forests, to identify miRs whose concomitant expression could generate an effective outcome predictor in CN-AML. To account for the effect of co-existing prognostic gene mutations, we included the European LeukemiaNet (ELN) risk group status in the random forest analyses. A total of 8 prognostic miRs were identified, 4 of which were also found to be prognostic in univariable analysis (underlined below), thus bringing the total number of unique prognostic miRs to 13: miR-511, miR-1193, miR-155, miR-4517, miR-3681, miR-2355, miR-628, miR-1266, miR-6715a, miR-1180, miR-6715b, miR-132, miR-146b. We used partitioning around medoids to divide our pts into clusters, based on the combined expression levels of the 13 prognostic miRs. Two such clusters were identified: cluster 1 comprised 173 pts and cluster 2 contained 108 pts. Regarding pretreatment and molecular features, pts in cluster 1 had lower percent of bone marrow blasts (P=.04), and had more frequently biallelic CEBPA mutations (P<.001) and less frequently internal tandem duplications of the FLT3 gene (P<.001), RUNX1 (P=.02) and WT1(P=.03) mutations than pts in cluster 2. In outcome analyses, pts in cluster 1 had a higher complete remission rate (CR; 91% vs 73%,P<.001) and a longer disease-free survival (DFS; 5-y rates 52% vs 16%, P<.001) and OS (5-y rates 60% vs 19%,P<.001). In multivariable analysis, cluster 1 status remained significantly associated with higher odds of achieving a CR (P=.001) and longer DFS (P<.001) and OS (P<.001), after adjusting for other covariates. Regarding accuracy of outcome prediction, our composite model had a concordance index of 0.687. When ranked according to importance for prognosis, miR-511 expression was the most significant determinant among the random forest model parameters. To evaluate the reproducibility of our findings, we performed analyses in the publicly available TCGA dataset (Ley et al. NEJM 2013;368:2059). Eighty-eight CN-AML pts with miR expression and survival data were available in the TCGA cohort. As TCGA pts are not classified according to ELN risk groups, we could not directly reproduce the random forest-based cluster analysis. However, a univariable analysis showed that miR-511 and miR-628 expression levels were also prognostic in the TCGA dataset. Next, we evaluated whether the identified prognostic miRs have functional relevance in AML. We focused on miR-511, which was the most important determinant of our outcome predictor and has not been previously studied in AML. Among 6 AML cell lines tested, MV4-11 had the most abundant expression of miR-511. Functional silencing of miR-511 in MV4-11 cells decreased both their viability (as measured by Annexin-PI staining and flow-cytometry, P=.004) and proliferative capacity (as measured by WST1 reagent degradation, P<.001). Conclusion: Unbiased profiling of miRs using smRNA-seq has identified a novel set of 13 miRs with prognostic significance in CN-AML. MiR expression-based cluster status independently associates with clinical outcome of CN-AML pts. Our preliminary in vitro experiments have shown that miR-511, whose association with prognosis was the strongest among the newly identified prognostic miRs, is functionally relevant in AML. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Powell:Rafael Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding; Janssen: Research Funding. Kolitz:Astellas: Research Funding; Boeringer-Ingelheim: Research Funding; Roche: Research Funding. Byrd:Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau.
