Gvhd Targets Organoid-Forming Biliary Epithelial Stem Cells Via a TGF-β-Dependent Manner

Abstract [Introduction] Graft-versus-host disease (GVHD) is a potentially life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). We and others have shown that GVHD targets adult tissue stem cells in the gut and skin, while a role of liver tissue stem cells in hepatic GVHD remains to be clarified. Biliary epithelial cells (BECs) are primary targets in hepatic GVHD and a single BEC stem cell gives rise to multipotent liver organoids (Cao W: Gastroenterology 2017). We studied the fate and role of BEC stem cells in experimental hepatic GVHD. [Methods] B6D2F1 recipients were lethally irradiated and transplanted with 5 x 10 6 splenocytes plus 5 x 10 6 bone marrow cells from allogeneic (B6) or syngeneic (B6D2F1) donors on day 0. Liver organoids were generated from the bile ducts isolated from the liver right lobe. [Results] Flowcytometric analyses of the liver demonstrated donor T cell infiltration in the liver within the first week after allo-HCT, followed by massive infiltration of monocytes and macrophages. Hepatic GVHD was characterized by apoptosis of BEC (Figure 1A), elevated expression of Mmp7, a biomarker of biliary injury (Figure 1B), and elevation of plasma levels of total bilirubin (Figure 1C). To evaluate fate of BEC stem cells in hepatic GVHD, we enumerated BEC-derived organoids generated from the right lobe of the liver isolated after allo-HCT. The organoid-forming BEC stem cells were profoundly reduced at later time point after allo-HCT (Figure 1D), while they persisted in syngeneic controls, indicating that GVHD targets BEC stem cells. Next, we explored the mechanism of BEC stem cell injury. Among the cytokines elevated in the liver after allo-HCT, we found that TGF-β , not IFN-γ or TNF-α, inhibited generation of liver organoids (Figure 1E). Furthermore, we found that liver infiltrating mononuclear cells isolated from the allogeneic livers, not from syngeneic livers, suppressed growth of liver organoids. This effect was abrogated by the addition of a TGF-β inhibitor, SB-431542 in culture. Among these cells, monocyte-derived macrophages, not Kupffer cells, demonstrated enhanced production of TGF-β after allo-HCT (Figure 1F), suggesting that TGF-β from inflammatory macrophages damaged BEC stem cells. Based on these findings, we next tested if TGF-β inhibition could protect BEC stem cells and ameliorate hepatic GVHD after allo-HCT. Admnistration of SB-431542 from day +14 to day +28 after allo-HCT significantly increased organoid-forming BEC stem cells, suppressed BEC apoptosis and Mmp7 expression, and mitigated jaundice on day +28 after allogeneic HCT, indicating that TGF-β inhibition is a novel therapeutic strategy against hepatic GVHD (Figure 1, G-J). [Conclusion] Our results for the first time demonstrated that hepatic GVHD targets BEC stem cells via a TGF-β-dependent manner. Mmp7 and organoid-forming capacity could be the biomarkers for hepatic GVHD. BEC stem-cell protection by TGF-β inhibition is a promising novel therapeutic strategy against hepatic GVHD. Figures1: (A) Proportion of cleaved caspase 3 (cCaspase3) + cells among the biliary epithelial cells (BECs) on day +28. (B) Total RNA extracted from the liver on day +28 was subjected to Q-PCR targeting Mmp7. (C) Plasma levels of total bilirubin at indicated time points. (D) The numbers of organoid derived from the right lobe of the liver at indicated time point are shown. (E) Absolute numbers of TGF-β producing Kupffer cells and macrophages (Mφ) in the liver on day +28 are shown. (F) Liver organoids were enumerated after incubation in the presence or absence of TGF-β for 4 days. (G-J) Allogeneic recipients were intraperitoneally injected with 5 mg/kg SB-431542 daily from day +14 to day +28 after allogeneic HCT, and recipient mice were sacrificed on day +28. Numbers of organoids derived from the right lobe of the livers (G), the proportion of cCaspase3 + BECs (H), relative expression of Mmp7 in the liver (I), and plasma levels of total bilirubin (J) are shown. *; p < .05, **; p < .01, ***; p < .005. Figure 1 Figure 1. Disclosures Teshima: CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Takeda Pharmaceutical Company: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis International AG: Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Gentium/Jazz Pharmaceuticals: Consultancy; Bristol Myers Squibb: Honoraria; Sanofi S.A.: Research Funding; TEIJIN PHARMA Limited: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fuji pharma CO.,Ltd: Research Funding; Pfizer Inc.: Honoraria; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin Co.,Ltd.: Honoraria, Research Funding; Astellas Pharma Inc.: Research Funding; Janssen Pharmaceutical K.K.: Other.

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A Comparison of Chemotherapy + G-CSF Versus Plerixafor (Mozobil®) + G-CSF for Stem Cell Mobilization In Patients with Multiple Myeloma Treated with Lenalidomide

Blood ◽

10.1182/blood.v116.21.2258.2258 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2258-2258

Author(s):

Tomer M Mark ◽

Adriana C Rossi ◽

Roger N Pearse ◽

Morton Coleman ◽

David Bernstein ◽

...

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Stem Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Yield ◽

High Dose ◽

Advisory Committees ◽

Cd34 Cells ◽

Stem Cell Collection

Abstract Abstract 2258 Background: Prior use of lenalidomide beyond 6 cycles of therapy in the treatment of multiple myeloma (MM) has been shown to negatively impact stem cell yield, but this phenomenon can be overcome with the addition of high-dose cyclophosphamide to standard G-CSF mobilization. We hypothesized that the use of plerixafor (Mozobil®) would compare similarly to chemotherapy in rescuing the ability to collect stem cells in lenalidomide-treated myeloma. Methods: We performed a retrospective study comparing the efficacy of plerixafor + G-CSF mobilization (PG) to chemotherapy + G-CSF (CG) (either high-dose cyclophosphamide at 3g/m2 or DCEP [4-day infusional dexamethasone/ cyclophosphamide/ etoposide/cisplatin]) in 49 consecutive stem cell collection attempts in patients with MM exposed to prior lenalidomide. The primary endpoint was the ability to collect sufficient stem cells for at least two transplants (minimum 5×106 CD34+ cells/kg), comparing results in terms of total exposure to lenalidomide and time elapsed from lenalidomide exposure until the mobilization attempt. The secondary endpoint was number of apheresis days required to meet collection goal. Resilts: Twenty-four patients underwent PG mobilization and twenty-five with CG (21 with G-CSF + cyclophosphamide, 4 with G-CSF+DCEP). The two groups did not differ in terms of total amount of lenalidomide exposure: median number of lenalidomide cycles for patients mobilized with PG was 6.5 (range 1.2–86.6), vs. 6 (range 2–21.6), for patients mobilized with CG (P = 0.663). The median time between mobilization and last lenalidomide dose was also similar between the two groups: 57.5 (range 12–462) days for PG vs. 154 (range 27–805) days for CG (P = 0.101). There was an equivalent rate of successful collection of 100% for PG and 96% for CG, P = 0.322. One patient failed collection in the CG group due to emergent hospitalization for septic shock during a period of neutropenia; no patient collected with PG had a serious adverse event that interrupted the collection process. Stem cell yield did not differ between the two arms (13.9 vs. 18.8 × 106 million CD34+ cells/kg for PG vs. CG respectively, P = 0.083). Average time to collection goal was also equal, with a median of time of 1 day required in both groups, (range 1–2 days for PG, 1–5 days for CG, P = 0.073). There was no relationship between amount of lenalidomide exposure and stem cell yield with either PG (P = 0.243) or CG (P = 0.867). Conclusion: A plerixafor + G-CSF mobilization schedule is equivalent in efficacy to chemotherapy + G-CSF in obtaining adequate numbers of stem cells for two autologous stem cell transplants in patients with MM exposed to lenalidomide; however, PG may be a less toxic approach than chemomobilization. Number of lenalidomide cycles has no impact on chances of stem cell collection success using either method. Disclosures: Mark: Celgene Corp: Speakers Bureau; Millenium Corp: Speakers Bureau. Zafar: Celgene Corp: Speakers Bureau. Niesvizky: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Millenium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding.

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Impact of Melphalan-Based Chemotherapy on Stem Cell Collection in Patients with Light Chain Amyloidosis

Blood ◽

10.1182/blood.v128.22.2187.2187 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2187-2187

Author(s):

Surbhi Sidana ◽

Nidhi Tandon ◽

Angela Dispenzieri ◽

Morie A. Gertz ◽

Francis K Buadi ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Board Of Directors ◽

Light Chain ◽

Research Funding ◽

Cell Yield ◽

Advisory Committees ◽

Prior Chemotherapy ◽

Cell Harvest ◽

Stem Cell Collection

Abstract Introduction: Melphalan based chemotherapy is commonly used for treatment of light chain amyloidosis (AL). Patients with AL often receive chemotherapy before autologous stem cell transplantation (ASCT) if they have high plasma cell burden or while awaiting ASCT. Melphalan is an alkylator and can affect bone marrow stem cells. Limited data is available on the effect of melphalan on stem cell mobilization in patients with amyloidosis. This study aims to identify the impact of melphalan therapy on collection of stem cells and ASCT in amyloidosis. Methods: All patients with AL seen at our institution within 90 days of diagnosis over a 10-year period (2006 to 2015) who underwent stem cell harvest were identified from an institutional database. Data pertaining to demographics, diagnosis, treatment, stem cell harvest and ASCT was extracted from the electronic medical records. Analysis was carried out by chi-square and Fisher's exact test for categorical variables and Kruskal-Wallis and Wilcoxon rank sum test for ordinal and continuous variables. Results: Three hundred and seventy two patients with AL who met the inclusion criteria were identified, of whom 10% (n=38) received melphalan based chemotherapy prior to harvesting, 28.5% (n=106) received non-melphalan based chemotherapy and 61.3% (n=228) received no chemotherapy prior to stem cell collection. Bortezomib based regimens were the most common (78%, n=83) non-melphalan based chemotherapy. All three groups were similar in terms of median age at diagnosis (59.1 years), median age at collection (59.4 years), gender distribution (59% males, n=221) and type of involved free light chain (FLC), with lambda being more common (72.2%, n=268). Patients who received melphalan-based chemotherapy had more cardiac (73.8% vs. 45.2% vs. 46.4%, p=0.005) and renal (84.2% vs. 50.9% vs. 68%, p=0.0002) involvement compared to other chemotherapy and no chemotherapy groups, respectively. In contrast, patients who received non-melphalan based chemotherapy had higher plasma cell burden (15% vs. 6% vs. 10%, p< 0.0001) and greater difference between involved and uninvolved FLC (44.2 mg/dL vs. 13.3 mg/dL vs. 13.2 mg/dL, p< 0.0001) compared to melphalan and no chemotherapy, respectively. Median duration of melphalan based chemotherapy was shorter at 54 days (34.5 to 79.5) or estimated 2 cycles compared to 101 days (60 to 135.5) or estimated 4 cycles (p=0.0019). Despite shorter duration of chemotherapy, total stem cell yield (million CD34/kg) was lower in patients who received melphalan based chemotherapy (5.54) compared to non-melphalan based chemotherapy (8.14) or no prior chemotherapy (7.94); p<0.0001. Similarly, day one stem cell yield (million CD34/kg) was the lowest in the melphalan group (2.71), followed by other chemotherapy group (3.63) and highest in no chemotherapy group (4.84); p<0.0001. This trend persisted for average stem cell yield per collection as illustrated in table 1. Filgrastrim (GCSF) alone was the most common mobilizing agent. However, patients with any chemotherapy prior to harvesting had higher utilization of plerixafor; 26.3% (n=10) in the melphalan group and 39.6% (n=42) in the non-melphalan group compared to 11.6% (n=27) if no prior chemotherapy (p<0.0001). However, no statistically significant difference was seen for melphalan vs. non-melphalan chemotherapy groups (p=0.44). In patients who underwent ASCT (85%, n=315), median stem cell dose (million CD34/kg) was different in the melphalan (3.66), non-melphalan (4.2) and no chemotherapy groups (4.44) (p=0.047), though the difference was not statistically significant amongst the 2 chemotherapy groups (p=0.34). There was also no difference in time to engraftment (table 1). Conclusions: Melphalan based chemotherapy, even if used for a short duration of time, significantly decreases both total stem cell yield and the yield on day one. It therefore has the potential to add to resource utilization with more collections needed. As much as possible, limited cycles of melphalan based chemotherapy or non-melphalan based treatment should be utilized in patients who are transplant eligible. Disclosures Dispenzieri: GSK: Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Jannsen: Research Funding; Celgene: Research Funding; pfizer: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kapoor:Amgen: Research Funding; Takeda: Research Funding; Celgene: Research Funding. Kumar:Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Glycomimetics: Consultancy; BMS: Consultancy; Sanofi: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Noxxon Pharma: Consultancy, Research Funding; Kesios: Consultancy.

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Combination of the Hedgehog Pathway Inhibitor LDE225 and Nilotinib Eliminates Chronic Myeloid Leukemia Stem and Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.1428.1428 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1428-1428 ◽

Cited By ~ 8

Author(s):

David A Irvine ◽

Bin Zhang ◽

Elaine K Allan ◽

Tessa L Holyoake ◽

Marion Dorsch ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Board Of Directors ◽

Colony Formation ◽

Research Funding ◽

Drug Control ◽

Advisory Committees ◽

Long Term Culture ◽

Short Term Culture

Abstract Abstract 1428 Poster Board I-451 Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder arising in a hematopoietic stem cell (HSC). CML is associated with expression of the Philadelphia chromosome (Ph) and its fusion gene product, BCR-ABL, a constitutively active tyrosine kinase. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib, all show impressive rates of complete cytogenetic response in chronic phase (CP) CML. However, the majority of responding CP CML patients have detectable BCR-ABL transcripts which might arise from a population of quiescent CML stem cells not effectively targeted by TKIs. Recent studies have indicated that the Hedgehog (Hh) pathway, a developmental pathway with roles in primitive and adult hematopoiesis, is activated in CML stem cells via upregulation of Smoothened (SMO), a seven-transmembrane domain receptor protein (Dierks et al, Cancer Cell 2008;14:238). We found that Gli1, a downstream target of Hh signalling, is significantly upregulated (6-fold) at the mRNA level in CD34+ enriched CP CML cells compared to normal CD34+ hematopoietic cells by Taqman quantitative RT-PCR. Thus, inhibition of SMO may be an effective therapeutic strategy to reduce the quiescent CML stem cell pool. LDE225 (Novartis Pharma) is a small molecule SMO antagonist which has recently entered Phase 1 clinical evaluation in patients with solid tumors. We assessed the efficacy of LDE225, alone and in combination with nilotinib, in primary CD34+ CML cells in vitro using short- and long-term culture techniques, including colony forming cell (CFC) assays with replates, long-term culture-initiating cell (LTC-IC) assays, CFSE-based flow cytometry to track cell division, Annexin/Viaprobe to measure apoptosis and Ki-67/7AAD cell cycle analysis. In short-term culture, incremental concentrations of LDE225 (0.5 - 1000nM) had no effect on total viable cell counts up to 12 days and did not inhibit cell proliferation as assessed by BrDU incorporation and CFSE staining. LDE225 did not increase apoptosis or alter the cell cycle profile of CD34+ CML cells compared to untreated controls. The lack of response in short-term culture experiments is explained by the stem cell selective nature of LDE225 which affects self-renewal and not proliferation or apoptosis. For long-term culture experiments, CD34+ CML cells were cultured for either 3 or 7 days in serum-free media supplemented with physiological growth factors (IL-3, IL-6, G-CSF, FLT-3 and SCF) with and without LDE225; cells were then washed and put into CFC assays with no added LDE225. Increasing concentrations of LDE225 did not significantly reduce colony read-out in the CFC assays. However, when the CFCs were serially replated there was a highly significant reduction in secondary colony formation and replating efficiency with increasing concentrations of LDE225 up to a plateau at 50nM during the initial 72 hour culture. In the first re-plate, colony formation was reduced by 38% with 5nM (P=0.07) and 62% with 50nM LDE225 (P=0.011; n=3) compared to a “no drug” control. Further reductions in colony formation were seen in second and subsequent replates. In further CFC replating experiments, after 3 and 7 days initial exposure to the combination of LDE225 10nM + nilotinib 5μM, replating efficiency was reduced by 50% (P<0.03) and 74%,(P<0.005) respectively (n=3). Single agent nilotinib resulted in a non-significant increase in colony formation and replating efficiency. In LTC-IC assays, compared to the “no drug” control, CD34+ CML cells showed increased colony formation in the nilotinib arm, indicating that, by inhibiting proliferation, nilotinib exerts a protective effect on CML stem cells (as shown previously for imatinib and dasatinib; Copland et al, Blood 2008;111:2843). The arm containing LDE225 + nilotinib showed a reduction in colony formation compared to both the nilotinib arm (85% reduction) and the “no drug” control (50% reduction). These results confirm the stem cell selectivity of LDE225. In conclusion, LDE225 targets CML stem cells and, in combination with nilotinib, represents a novel therapeutic strategy for targeting both the primitive quiescent CML stem cell population and the bulk CML progenitor population. In vivo studies in murine models of CML are ongoing to help further evaluate the clinical potential of LDE225 in combination with nilotinib. Disclosures Holyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Dorsch: Novartis: Employment. Manley: Novartis: Employment. Bhatia: Novartis: Consultancy. Copland: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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A Novel TNF-Alpha Antibody Based Therapeutic Approach to Target Leukemic Stem Cells in Bcr-Abl Disease

Blood ◽

10.1182/blood.v126.23.15.15 ◽

2015 ◽

Vol 126 (23) ◽

pp. 15-15

Author(s):

Oliver Herrmann ◽

Claudia Schubert ◽

Tom Luedde ◽

Till Braunschweig ◽

Steffen Koschmieder ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Board Of Directors ◽

Cell Population ◽

Research Funding ◽

Stem Cell Population ◽

Tnf Alpha ◽

Spleen Weight ◽

Leukemic Stem Cells ◽

Advisory Committees

Abstract Chronic myeloid leukemia (CML) reflects the biology of a stem cell driven neoplasm. The cytogenetic marker t(9;22) gives rise to Bcr-Abl protein that activates various signaling pathways resulting in increased proliferation and differentiation, protection from apoptosis and altered adhesion properties of CML cells. The implementation of Bcr-Abl tyrosine kinase inhibitors (TKI) has greatly improved therapy outcome of CML patients. However, more than one third of newly diagnosed CML patients develops primary or secondary resistance mostly due to Bcr-Abl mutations or intolerance to TKIs. Moreover, even in patients without mutations, mechanisms such as low persisting Bcr-Abl levels in patients on TKI and/or a lack of oncogene addiction have been described to induce CML stem cell persistence. We have previously demonstrated Bcr-Abl independent stem cell persistence using inducible SCLtTA/Bcr-Abl mice (BLOOD 2010, 115:3185; BLOOD 2012, 119:1501), and recent data suggest an important role for TNF-alpha in MPN stem cell persistence (BLOOD 2011, 118:6392), including CML (BLOOD 2013, 112:3335). Therefore, we have now interrogated our mouse model for a novel stem cell directed therapeutic approach, using pharmacologic inhibition of TNF-alpha. Analysis of TNF-alpha expression by qRT-PCR in Bcr-Abl infected 32Dcl3 and Ba/F3 cells revealed elevated cytokine expression upon Bcr-Abl transduction. Imatinib treatment of 32Dcl3:Bcr-Abl cells reverted elevated TNF-alpha levels suggesting that TNF-alpha upregulation is kinase mediated in this cell line. ChIP-Seq of TKI treated vs non-treated 32Dcl3:Bcr-Abl cells showed H3K9 acetylation of the TNF-alpha promoter in untreated cells, which decreased upon Bcr-Abl inhibition. Subsequently, we analyzed TNF-alpha expression in lin-;Sca-1+;c-kit+ (LSK) cells isolated from SCLtTA/Bcr-Abl mice that had been induced to express Bcr-Abl for 3 weeks. Leukemic LSK cells showed again significantly increased TNF-alpha expression. Interestingly, TNF-alpha RNA levels were not altered in total bone marrow (BM) cells isolated from transplanted leukemic Bcr-Abl mice compared to controls, suggesting a stem cell specific mechanism regulating TNF-alpha expression in vivo. Next, we tested the therapeutic effect of TNF-alpha inhibition combined with TKI therapy. We applied the chimeric antibody infliximab that blocks soluble as well as membrane-bound TNF-alpha. We transplanted 1.5x105 wildtype (wt) or Bcr-Abl BM cells expressing CD45.1 into 10 Gy irradiated CD45.2+ wt recipients (wt n=4; Bcr-Abl n=24) and allowed these cells to engraft and expand for 2 weeks. We then either treated Bcr-Abl transplanted mice with nilotinib alone (50mg/kg, daily) or combined the TKI therapy with infliximab (10mg/kg, weekly i.v.). As controls, we either treated Bcr-Abl transplanted mice with vehicle alone (daily) or together with IgG (10mg/kg, i.v., weekly). Wt recipients remained untreated. After 2.5 weeks of therapy, we sacrificed all mice for analyses. Spleen weight of vehicle and vehicle+IgG treated mice were increased compared to nilotinib and nilotinib+infliximab treated recipients (Table 1). Table 1. Analyses of mice that were subjected to TNF-alpha and Bcr-Abl inhibition wt Bcr-Abl vehicle vehicle + IgG nilotinib nilotinib + infliximab spleen weight (mg) 73 ± 5 274 ± 63 280 ± 55 125 ± 5 185 ± 31 % of lin- cells 4.8 ± 1.1 31.1 ± 11.0 25.0 ± 14.1 3.4 ± 1.0 3.1 ± 1.4 % of LSK/CD45.1+ cells 5.7 ± 0.7 41.2 ± 5.9 31.6 ± 11.7 20.3 ± 10.8 7.2 ± 3.4 FACS analyses s that lin- cells were significantly elevated in vehicle and vehicle+IgG treated Bcr-Abl recipients compared to wt mice. Nilotinib and nilotinib+infliximab therapy decreased lin- cells to 3.4% and 3.1% respectively. As expected, leukemic CD45.1+ LSK cells were significantly increased in vehicle (7-fold) and vehicle+IgG (5.5-fold) treated mice compared to CD45.1+ wt LSK cells. Nilotinib treatment decreased the elevated levels of CD45.1+ LSK cells compared to wt controls to 3.6-fold but combining TKI+infliximab decreased the leukemic stem cell population further to 1.3-fold difference compared to wt mice. In conclusion, we show that TNF-alpha expression is elevated in Bcr-Abl positive leukemic stem cells and this cell population is significantly reduced by combining TKI therapy with an anti TNF-alpha approach. Our data reveal that this combinational therapy is a powerful tool to target the TKI resistant stem cell population in CML. Disclosures Koschmieder: Janssen Cilag: Other: Travel reimbursement for scientific conferences; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences; Sanofi: Membership on an entity's Board of Directors or advisory committees; Baxalta/CTI: Membership on an entity's Board of Directors or advisory committees; Novartis Foundation: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences, Research Funding. Brümmendorf:Ariad: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria. Schemionek:Novartis Foundation for Therapeutic Research: Research Funding. Off Label Use: Infliximab used in CML mouse model.

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Unexpected Toxicities When Nivolumab Was Given after Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-123887 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1956-1956

Author(s):

Amy Wang ◽

Justin Kline ◽

Wendy Stock ◽

Satyajit Kosuri ◽

Andrew S. Artz ◽

...

Keyword(s):

Clinical Trial ◽

Overall Survival ◽

Stem Cell ◽

Adverse Events ◽

Maintenance Therapy ◽

Board Of Directors ◽

Research Funding ◽

Checkpoint Inhibitors ◽

Advisory Committees ◽

Relapsed Disease

Background:Treatment options are limited for patients (pts) with hematologic malignancies who relapse after allogeneic stem cell transplantation (allo-SCT). We hypothesized that checkpoint inhibitors may offer a novel approach for maintaining remission after allo-SCT. Data from pre-clinical studies have suggested a potential role for PD-1/PD-L1 inhibitors in acute myeloid leukemia (AML) (Zhang et al., Blood 2009), so it is possible that immunomodulation with checkpoint inhibitors could stimulate the donor anti-leukemia immune response and prevent disease relapse. However, the safety of checkpoint blockade early after allografting remains to be established. Methods:We conducted a pilot study to assess the tolerability and efficacy of Nivolumab, a PD-1 inhibitor, as maintenance therapy after allo-SCT (NCT02985554). Pts were eligible if they were post allo-SCT without evidence of relapse or active graft-vs-host disease (GVHD) or history of prior greater than stage I skin acute GVHD. Nivolumab was to be administered intravenously at 1mg/kg every 2 weeks for 4 doses followed by dosing every 12 weeks. Treatment started 4 weeks after routine immunosuppression was discontinued until 2 years after the transplant. The primary objective was to determine the tolerability of Nivolumab on this schedule. Secondary objectives were evaluation of adverse events, relapse, and overall survival. Results:Four pts were enrolled from December 2017 through November 2018. (Table 1)All pts experienced immune-related adverse events (irAE) from Nivolumab, and 2 (50%) pts experienced serious adverse events. (Table 2)One pt developed grade (G) 4 neutropenia soon after the first dose. (Figure 1)The absolute neutrophil count nadired at 20 cells/µL, at which point pegfilgrastim was administered. An interim bone marrow biopsy (BMBx) confirmed no evidence of relapsed disease. Full neutrophil recovery occurred approximately 3 months after the initial dose, and no subsequent toxicities occurred. Another pt developed G3 autoimmune encephalopathy concurrently with G2 transaminitis and G2 thrombocytopenia after one dose of Nivolumab. (Figure 2)Intravenous methylprednisolone (1mg/kg daily for 3 days) and immunoglobulin (2g/kg in 4 divided doses) were administered, followed by a 7-week steroid taper with full resolution of symptoms. Relapsed disease was ruled out by a BMBx. A third pt developed G2 skin rash approximately 10 days after the first dose of Nivolumab. Skin biopsy demonstrated drug hypersensitivity reaction vs GVHD, and the pt was treated with a 3-week prednisone course (starting at 1mg/kg followed by a taper). A mild flare recurred 2 weeks later, which was treated with topical steroids only. However, Nivolumab was not resumed. The fourth pt developed G2 elevated TSH approximately 2 months into therapy and after 4 doses of Nivolumab. Thyroid hormone replacement was initiated with subsequent symptom improvement and normalization of TSH over a 4-month period. As a result of these unexpected severe toxicities, the study was closed to further enrollment, and further Nivolumab administration ceased. Thus far, one pt (#1) relapsed after a total remission duration of 530 days; the remission duration after starting Nivolumab was 318 days. One pt has mild chronic skin GVHD. All 4 patients remain alive with a median overall survival of 2.3 years (range, 1.9-4.7). Conclusions:Even at low doses, the use of Nivolumab as maintenance therapy in the post allo-SCT setting was not tolerable at the current dosing and schedule due to an unexpected number of high grade irAEs. Additional studies of dose and timing after allo-SCT are needed to improve safety and tolerability, in conjunction with correlative studies to better understand the immunomodulatory processes in the post-transplant setting. Disclosures Kline: Merck: Honoraria; Merck: Research Funding. Stock:Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; UpToDate: Honoraria; Research to Practice: Honoraria. Artz:Miltenyi: Research Funding. Larson:Agios: Consultancy; Novartis: Honoraria, Other: Contracts for clinical trials; Celgene: Consultancy. Riedell:Novartis: Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Speakers Bureau; Kite/Gilead: Honoraria, Research Funding, Speakers Bureau. Bishop:CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Juno: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Liu:Arog: Other: PI of clinical trial; BMS: Research Funding; Agios: Honoraria; Novartis: Other: PI of clinical trial; Karyopharm: Research Funding. OffLabel Disclosure: Nivolumab used as maintenance therapy in the post-transplant setting

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Multicenter Analysis of Treatment and Outcomes for Patient with TP53 Mutated AML in the Era of Novel Therapies; Significant Impact of Allogeneic Stem Cell Transplantation on Survival

Blood ◽

10.1182/blood-2021-147639 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 797-797

Author(s):

Talha Badar ◽

Mark R. Litzow ◽

Rory M. Shallis ◽

Jan Philipp Bewersdorf ◽

Antoine Saliba ◽

...

Keyword(s):

Stem Cell ◽

High Risk ◽

Stem Cell Transplantation ◽

Board Of Directors ◽

Research Funding ◽

Univariate Analysis ◽

Advisory Board ◽

Novel Therapies ◽

Advisory Committees ◽

Tp53 Mutations

Abstract Background: TP53 mutations occur in 10-20% of patients with AML, constitute high-risk disease as per ELN criteria, and confer poorer prognosis. Venetoclax combination therapies and CPX-351 were recently approved for AML treatment and lead to improved outcomes in subsets of high-risk AML, however the most effective approach for treatment of TP53-mutated (m) AML remains unclear. In this study we explored the clinical outcome of TP53m AML patients treated over the last 8 years as novel therapies have been introduced to our therapeutic armamentarium. Methods: We conducted a multicenter observational study in collaboration with 4 U.S. academic centers and analyzed clinical characteristics and outcome of 174 TP53m AML patients diagnosed between March 2013 and February 2021. Mutation analysis was performed on bone marrow specimens using 42, 49, 199, or 400 gene targeted next generation sequencing (NGS) panels. Patients with an initial diagnosis of AML were divided into 4 groups (GP) based on the progressive use of novel therapies in clinical trials and their approvals as AML induction therapy during different time periods: 2013-2017 (GP1, n= 37), 2018-2019 (GP2, n= 53), 2019-2020 (GP3, n= 48) and 2020-2021 (GP4, n= 36) to analyze difference in outcome. Results: Baseline characteristics were not significantly different across different GP, as shown in Table 1. Median age of patients was 68 (range [R], 18-83), 65 (R, 29-88), 69 (R, 37-90) and 70 (R, 51-97) years in GP1-4, respectively (p=0.40). The percentage of patients with de novo AML/secondary AML/therapy-related AML in GP1-4 was 40/40/20, 36/29/24, 37.5/37.5/25 and 28/52/20, respectively (p=0.82). The proportion of patients with complex cytogenetics (CG) was 92%, 89%, 96% and 94% in GP1-4, respectively (p=0.54). The median TP53m variant allele frequency (VAF) was 48% (range [R], 5-94), 42% (R, 5-91), 45% (R, 10-94) and 60% (R, 8-82) in GP1-4, respectively (p=0.38). Four (11%), 13 (24.5%), 10 (21%) and 9 (25%) patients had multiple TP53 mutations in GP1-4, respectively (p=0.33). The proportion of patients who received 3+7 (30%, 16%, 6% & 8%; p=0.01), HMA only (11%, 18%, 2% & 8%; p=0.06), venetoclax-based (2.5%, 12%, 48%, & 61%; p <0.01) and CPX-351 induction (16%, 40%, 28% & 5%; p<0.001) were varied in GP1-4, respectively. The rate of CR/CRi was 22%, 26%, 28% and 18% in GP1-4, respectively (p=0.63). Treatment related mortality during induction was observed in 3%, 7%, 10% and 17% of patients in GP1-4, respectively (p=0.18). Overall, 28 (16%) patients received allogeneic hematopoietic stem cell transplantation (alloHCT) after induction/consolidation: 22%, 15%, 17% and 11% in GP1-4, respectively (p=0.67). In subset analysis, there was no difference in the rate of CR/CRi with venetoclax-based regimens vs. others (39% vs 61%, p=0.18) or with CPX-351 vs. others (25% vs 75%, p=0.84). The median progression-free survival was 7.7, 7.0, 5.1 and 6.6 months in GP1-4, respectively (p=0.60, Fig 1A). The median overall survival (OS) was 9.4, 6.1, 4.0 and 8.0 months in GP1-4, respectively (p=0.29, Fig 1B). In univariate analysis for OS, achievement of CR/CRi (p<0.001) and alloHCT in CR1 (p<0.001) associated with favorable outcome, whereas complex CG (p=0.01) and primary refractory disease (p<0.001) associated with poor outcome. Multiple TP53 mutations (p=0.73), concurrent ASXL1m (p=0.86), extra-medullary disease (p=0.92), ≥ 3 non-TP53m mutations (p=0.72), TP53m VAF ≥ 40% vs. < 40% (p=0.25), induction with CPX-351 vs. others (p=0.59) or venetoclax-based regimen vs. others (p=0.14) did not show significance for favorable or poor OS in univariate analysis. In multivariable analysis, alloHCT in CR1 (hazard ratio [HR]=0.28, 95% CI: 0.15-0.53; p=0.001) retained an association with favorable OS and complex CG (HR 4.23, 95%CI: 1.79-10.0; p=0.001) retained an association with dismal OS. Conclusion: We present the largest experience with TP53m AML patients analyzed by NGS. Although outcomes were almost universally dismal, alloHCT appears to improve the long-term survival in a subset of these patients. Effective therapies are warranted to successfully bridge patients to alloHCT and to prolong survival for transplant ineligible patients. Figure 1 Figure 1. Disclosures Badar: Pfizer Hematology-Oncology: Membership on an entity's Board of Directors or advisory committees. Litzow: Omeros: Other: Advisory Board; Pluristem: Research Funding; Actinium: Research Funding; Amgen: Research Funding; Jazz: Other: Advisory Board; AbbVie: Research Funding; Astellas: Research Funding; Biosight: Other: Data monitoring committee. Shallis: Curis: Divested equity in a private or publicly-traded company in the past 24 months. Goldberg: Celularity: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aprea: Research Funding; Arog: Research Funding; DAVA Oncology: Honoraria; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Prelude Therapeutics: Research Funding; Aptose: Consultancy, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Atallah: BMS: Honoraria, Speakers Bureau; Takeda: Consultancy, Research Funding; Amgen: Consultancy; Abbvie: Consultancy, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Research Funding. Foran: revolution medicine: Honoraria; gamida: Honoraria; bms: Honoraria; pfizer: Honoraria; novartis: Honoraria; takeda: Research Funding; kura: Research Funding; h3bioscience: Research Funding; OncLive: Honoraria; servier: Honoraria; aptose: Research Funding; actinium: Research Funding; abbvie: Research Funding; trillium: Research Funding; sanofi aventis: Honoraria; certara: Honoraria; syros: Honoraria; taiho: Honoraria; boehringer ingelheim: Research Funding; aprea: Research Funding; sellas: Research Funding; stemline: Research Funding.

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Outcome of Autologous Stem Cell Transplantation in Waldenström's Macroglobulinemia

Blood ◽

10.1182/blood-2018-99-116496 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2149-2149

Author(s):

Romil Patel ◽

Neeraj Y Saini ◽

Ankur Varma ◽

Omar Hasan ◽

Qaiser Bashir ◽

...

Keyword(s):

Overall Survival ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Board Of Directors ◽

Cell Transplantation ◽

Research Funding ◽

Advisory Committees ◽

First Remission ◽

Relapsed Disease

Abstract Introduction: The role of autologous hematopoietic stem cell transplantation (auto-HCT) in the management of patients with Waldenström Macroglobulinemia (WM), a rare, indolent lymphoma, has not been established. We had previously published our experience with auto-HCT in a small cohort of WM patients1. Here, we present an updated analysis of auto-HCT with a larger cohort of WM patients. Methods and study population: The study cohort was comprised of 29 patients who underwent high-dose chemotherapy and auto-HCT at MD Anderson Cancer Center (MDACC). The Kaplan-Meier method was used to create survival curves. Overall survival (OS) was defined as the duration from date of transplant to death or last date of follow-up in living patients. Progression-free survival (PFS) was defined as the duration from date of transplant to either progressive disease or death, whichever occurred first. Results: Median age at auto-HCT was 60 (range, 43-75 years). Eight patients (28%) had concurrent light chain amyloidosis (AL). Of the five patients who had MYD88 testing completed, 3 were positive for the MYD88 mutation. Additionally, of these 3 patients, 2 were also positive for CXCR4 mutation. Patients received a median of 2 lines (range 1-6) of therapy prior to auto-HCT; 3(10%) patients had primary refractory disease, 8(28%) were in first remission, and 18 (62%) had relapsed disease. Median time from transplant to last follow-up for the surviving patients was 5.3 years. Preparative regimens received by the patients were: Melphalan (n=20), BEAM-R (n=2), Busulfan/Melphalan (n=1), Cyclophosphomaide/Etoposide/total body irradiation (n=1), Thiotepa/Busulfan/Cyclophosphamide (n=1), and Carmustine/Thiotepa (n=1). Three patients further went on to receive allogeneic transplant either after relapse from auto-HCT or due to disease transformation to aggressive lymphoma. Twenty-eight patients achieved engraftment with a median time to neutrophil engraftment of 11 days (range, 10-15 days). One patient suffered primary graft failure due to progression of disease and died 84 days after transplant. Non-relapse mortality was 3.4% at 1 year. All patients were eligible for response evaluation. The median OS from diagnosis was 12.2 years. Overall response rate was 96%: complete response (n=8, 27.6%), very good partial response (n=5, 17.3%), partial response (n=15, 51.7%), and progressive disease (n=1, 3.4%). PFS and OS at 5 years were 43.3% and 62.9%, respectively. Median PFS and OS from auto-HCT were 4.1 and 7.3 years (Fig. 1A). The median OS from auto-HCT in first remission + primary refractory and relapsed disease was 8.2 years and 4.1 years, respectively.16 patients were alive at the time of censoring while 13 patients had died. Causes of death include relapsed disease (n=6), secondary malignancy (n=2), infection (n=1), chronic graft-versus-host disease (n=1), and unknown (n=3). 8 patients (28%) were positive for concurrent AL amyloidosis. The sites of amyloid involvement were kidneys (n=2), lungs (n=1), bone marrow (n=1), heart(n=1), lymph nodes(n=1), gastrointestinal tract (n=1) and subcutaneous fat aspirate(n=5). The median overall survival for patients with amyloid involvement (n=8) was 12 years. On univariate analyses, the number of chemotherapy regimens prior to transplant (≤ 2 vs >2 lines) was the strongest predictor of overall survival (p=0.03, HR 0.3, CI: 0.09-0.9, log-rank) and PFS (p=0.001, HR 0.24, CI: 0.07-0.85, log-rank). The median PFS in patients with ≤ 2 lines and > 2 lines of therapy was 71 months versus 19 months, respectively (Fig. 1B). Conclusion: Auto-HCT is safe and feasible in selected patients with WM, with a high response rate and durable remission even in patients with relapsed or refractory disease. References: Krina Patel et.al. Autologous Stem Cell Transplantation in Waldenstrom's Macroglobulinemia. Blood 2012 120:4533; Disclosures Thomas: Celgene: Research Funding; Bristol Myers Squibb Inc.: Research Funding; Acerta Pharma: Research Funding; Array Pharma: Research Funding; Amgen Inc: Research Funding. Lee:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies Corporation: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai Biopharmaceuticals: Consultancy; Takeda Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Orlowski:Takeda: Consultancy; Celgene: Consultancy; Spectrum Pharma: Research Funding; Janssen: Consultancy; Kite Pharma: Consultancy; Sanofi-Aventis: Consultancy; BioTheryX: Research Funding; Amgen: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy. Champlin:Otsuka: Research Funding; Sanofi: Research Funding. Patel:Poseida Therapeutics, Inc.: Research Funding; Takeda: Research Funding; Abbvie: Research Funding; Celgene: Research Funding.

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BCX9930, a Potent, Selective, Oral Factor D Inhibitor, Demonstrates Proof-of-Concept As Monotherapy in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Blood ◽

10.1182/blood-2020-138838 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 14-15

Author(s):

Austin Kulasekararaj ◽

Jacques Le Roux Malherbe ◽

Andrew McDonald ◽

Melanie Cornpropst ◽

Phil Collis ◽

...

Keyword(s):

South Africa ◽

Board Of Directors ◽

Research Funding ◽

Total Bilirubin ◽

Proof Of Concept ◽

Advisory Committees ◽

Clone Size ◽

The Past ◽

History Of ◽

Pre Treatment

INTRODUCTION: PNH, a rare, chronic, life-threatening disease, is characterized by hemolytic anemia due to uncontrolled activity of the complement alternative pathway (AP), bone marrow failure, and thrombosis. Inhibition of C5 by intravenously administered eculizumab and ravulizumab reduces intravascular hemolysis, but PNH red blood cells (RBCs) become opsonized and susceptible to extravascular hemolysis (Risitano et al, Blood 2009). Only approximately half of PNH patients become transfusion independent with eculizumab treatment (Hillmen et al, NEJM 2006). BCX9930 is a potent, selective, orally administered inhibitor of complement factor D. Inhibition of factor D may prevent both intravascular and extravascular hemolysis in PNH. In healthy subjects, BCX9930 showed linear pharmacokinetics and dose-related AP suppression, and was safe and generally well-tolerated over a wide dose range. Here we describe safety and laboratory data establishing proof-of-concept for BCX9930 monotherapy in PNH patients in Study BCX9930-101 (NCT04330534). METHODS: Ongoing Study BCX9930-101 includes an open-label, dose-ranging evaluation of BCX9930 in PNH subjects who may either be naïve to C5 inhibitors (and receive BCX9930 as monotherapy) or have an incomplete treatment response to eculizumab or ravulizumab (with BCX9930 added to existing treatment). Up to 4 sequential cohorts each use a forced titration design for the first 28 days (Figure 1). Subjects enrolled in South Africa can participate in an individualized 48-week extension if they derive benefit at Day 28. Clinical benefit from BCX9930 is evaluated using laboratory monitoring and symptom assessment. Safety and tolerability are evaluated via clinical and laboratory monitoring, causality of adverse events is assessed by investigators, and the study is overseen by an independent Data Monitoring Committee. Data from Cohort 1 through 28 days is reported; data from the extension and subsequent cohorts will be subsequently summarized as available. RESULTS: To date, four C5 inhibitor naïve PNH subjects in South Africa have enrolled in Cohort 1. These subjects had PNH for a median of 4.5 years; 2 subjects had a history of transfusions in the past year; 1 subject each had a history of aplastic anemia or major thrombosis. Pre-treatment lactate dehydrogenase (LDH), total bilirubin, hemoglobin (Hb), reticulocyte count, and RBC PNH Type III clone size ranged from 3.7-11.1 × ULN, 0.61-3.3 mg/dL, 6.1-11.6 g/dL, 0.13-0.29 × 106/µL, and 41.4%-88.6% respectively. Treatment over 28 days with 50 mg twice daily (BID; Days 1-14) and 100 mg BID (Days 15-28) of BCX9930 produced dose-dependent, clinically meaningful improvements across hemolysis biomarkers (Figure 2). Decreases were observed in LDH (4/4), reticulocytes (4/4), and total bilirubin (2/2 subjects with elevated pre-treatment values). Increases were observed in Hb (3/4) and PNH RBC clone size (4/4). One subject showed an initial response to BCX9930 50 mg BID, followed by worsening indicators of hemolysis temporally associated with an upper respiratory tract infection (URTI; onset on Day 7). With an increase in dose to 100 mg BID and resolution of the URTI, LDH and reticulocytes fell and Hb rose. All four subjects reported one or more PNH-associated symptoms, including hemoglobinuria, jaundice, fatigue, erectile dysfunction, headache and abdominal pain, prior to enrollment. With the exception of one subject with persistent hemoglobinuria, all symptoms resolved by Day 28 on BCX9930. Three subjects experienced moderate headache that resolved in < 3 days after initiating BCX9930. One subject developed a rash during treatment with amoxicillin for an URTI; the rash resolved while continuing BCX9930 dosing. One subject on concomitant chronic corticosteroids and azathioprine had an unrelated fatal serious adverse event of disseminated varicella during the study extension. Based on review of safety data, Cohort 2 opened at doses of 200 mg BID and 400 mg BID and, in the 3 subjects who continued into the extension, the dose was titrated to ≥ 200 mg BID. CONCLUSIONS: Oral BCX9930 elicited rapid changes in laboratory parameters indicative of reduced hemolysis and clinical benefit and was safe and generally well-tolerated over a 28-day dosing interval. These interim results establish proof of concept for monotherapy with BCX9930 in the treatment of C5-inhibitor naïve PNH patients and support evaluation of higher doses. Disclosures Kulasekararaj: Alexion:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau;Ra Pharma:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau;BioCryst Pharmaceuticals, Inc.:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;Apellis:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau;Roche:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Novartis:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau;Celgene:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau.Malherbe:Key Oncologics:Honoraria, Other: Conference sponsor;Novartis:Other: Conference sponsor;Astellas:Honoraria, Other: Conference sponsor;Takeda:Consultancy;Acino:Honoraria;Shire:Other: Conference sponsor;BioCryst Pharmaceuticals, Inc.:Consultancy;Janssen:Consultancy, Honoraria, Other: Conference sponsor;Roche:Honoraria, Other: Conference sponsor.McDonald:venetoclax advisory board in South Africa (in CLL context):Consultancy;Alberts Cellular Therapy:Current Employment.Cornpropst:BioCryst Pharmaceuticals, Inc.:Current Employment.Collis:BioCryst Pharmaceuticals, Inc.:Current Employment.Davidson:BioCryst Pharmaceuticals, Inc.:Current Employment.Chen:BioCryst Pharmaceuticals, Inc.:Current Employment.Tower:BioCryst Pharmaceuticals, Inc.:Current Employment.Gesty-Palmer:BioCryst Pharmaceuticals, Inc.:Current equity holder in publicly-traded company, Ended employment in the past 24 months.Sheridan:BioCryst Pharmaceuticals, Inc.:Current Employment.Risitano:Alexion:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau;Alnylam:Research Funding;Novartis:Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau;Pfizer:Speakers Bureau;Achillion:Membership on an entity's Board of Directors or advisory committees;Apellis:Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Biocryst:Membership on an entity's Board of Directors or advisory committees;RA pharma:Research Funding;Amyndas:Consultancy;Samsung:Membership on an entity's Board of Directors or advisory committees;Roche:Membership on an entity's Board of Directors or advisory committees;Jazz:Speakers Bureau.

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Clonal Tracking By Whole Genome Sequencing Permits Comprehensive Mapping of the Genomic Landscape in Pre- and Post-Gene Therapy Sickle Cell Patients

Blood ◽

10.1182/blood-2021-152262 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 559-559

Author(s):

Alyssa H. Cull ◽

Michael Spencer Chapman ◽

Marioara Ciuculescu ◽

Emily Mitchell ◽

Myriam Armant ◽

...

Keyword(s):

Gene Therapy ◽

Stem Cell ◽

Board Of Directors ◽

Sickle Cell ◽

Research Funding ◽

Phylogenetic Trees ◽

Insertion Site ◽

Advisory Board ◽

Advisory Committees ◽

Genomic Landscape

Abstract Recent advances in clonal stem cell tracking strategies have enabled interrogation of unperturbed human hematopoiesis. Whole genome sequencing (WGS) can be used to map the clonal dynamics of hematopoietic stem and progenitor cells (HSPCs) by employing spontaneous somatic mutations as unique clonal tags (Lee-Six et al., Nature, 2018). These tags allow for retrospective analysis of individual stem cell clones and the construction of phylogenetic trees mapping out stem cell relatedness, with mutations being acquired in a near-linear fashion over the course of an individual's life. The unprecedented level of information obtained in these studies is particularly well-suited to understanding genomic changes in gene therapy trials aimed at curing diseases such as sickle cell disease (SCD). In addition to mapping relatedness between stem cells, sequencing data can be used to better define mutational signatures for HSPC clones that have been successfully gene-modified as well as those that lack an integrated copy of the therapeutic vector. Given this method's ability to identify low frequency mutations in individual HSPC clones, mutations with extremely low variant allele frequencies can be detected much more readily than through traditional bulk sequencing approaches, something that is particularly relevant given recent safety concerns in some SCD gene therapy trials. In this study, we have mapped the clonal dynamics of HSPCs obtained from pre- and post-gene therapy samples from 4 SCD patients who have undergone autologous gene therapy performed using a BCL11A shmiR lentivirus vector (NCT 03282656, 12-36 months follow-up). HSPCs from mobilized peripheral blood (pre-gene therapy), bone marrow aspirates (both pre- and post-gene therapy) or unmobilized peripheral blood (post-gene therapy) were expanded as single clones and 1508 individual colonies were then sequenced using WGS to an average sequencing depth of 12.3x. Initial results indicate that the mean mutation burden per cell in a pre-gene therapy sample is elevated for some patients compared to what would be expected based on patient age in similar studies. In pre-gene therapy samples, the structure of the phylogenetic trees appeared to be highly polyclonal, indicating that there were no significant clonal expansion events prior to gene therapy. In one patient where we undertook extensive profiling, approximately 15-20 excess mutations per HSPC were observed across the entire genome 24 months after transplantation, presumably acquired as a consequence of gene therapy and/or reconstitution post-transplantation, which is equivalent to approximately one year of normal ageing without a transplantation intervention. However, no clonal expansions or driver mutations were identified at this 24 month follow-up timepoint, suggesting that no strong selective advantage or pre-leukemic events were present prior to or following the gene therapy protocol. Extending this approach to a wider range and larger number of patients will allow for comprehensive mapping of the genomic landscape and clonal evolution of stem cells in sickle cell patients and will also set the stage for improved assessment of safety and potential leukemia-initiating events in the context of gene therapy. Disclosures Esrick: bluebird bio: Consultancy. Williams: bluebird bio: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Analysis Advisory Board, Patents & Royalties: BCH licensed certain IP relevant to hemoglobinopathies to bluebird bio. The current license includes the potential for future royalty/milestone income. Bluebird has indicated they will not pursue this as a clinical program and BCH is negotiating return of, Research Funding; BioMarin: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Advisory Board; Beam Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Emerging Therapy Solutions: Membership on an entity's Board of Directors or advisory committees, Other: Chief Scientific Chair; Geneception: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Alerion Biosciences: Other: Co-founder (now licensed to Avro Bio, potential for future milestones/royalties); Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Novartis ETB115E2201 (eltrombopag in aplastic anemia). Advisory fees donated to NAPAAC.; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Membership on a safety advisory board (SAB): SAB position ended 05/20/2021. Co-founder , Patents & Royalties: Potential for future royalty/milestone income, X-SCID. Provided GMP vector for clinical trial, Research Funding. Campbell: Mu Genomics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Kent: STRM.bio: Research Funding.

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A Phase II Single Arm Study of Nivolumab As Maintenance Therapy after Autologous Stem Cell Transplantation in Patients with Hodgkin Lymphoma at Risk of Relapse or Progression

Blood ◽

10.1182/blood-2021-148139 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2455-2455

Author(s):

Carlos Bachier ◽

Henning Schade ◽

Behyar Zoghi ◽

Aravind Ramakrishnan ◽

Nirav N. Shah

Keyword(s):

Stem Cell ◽

High Risk ◽

Maintenance Therapy ◽

Board Of Directors ◽

Hodgkin Lymphoma ◽

Research Funding ◽

Residual Disease ◽

Refractory Disease ◽

Advisory Committees ◽

Extranodal Disease

Abstract Introduction: Autologous stem cell transplants (ASCT) are standard of care for patients with primary refractory or recurrent Hodgkin lymphoma (HL). While transplant results in cure for some patients, others relapse and succumb from their disease. Studies have found high expression of programmed death ligand 1 (PD-L1) in HL cells. The anti-PD-1 monoclonal antibody, nivolumab, has been safe and efficacious in the treatment of relapsed, refractory HL (Ansell et al. 2015). We evaluated the safety and efficacy of nivolumab maintenance therapy post-ASCT in high risk for relapse Hodgkin disease. Methods: Patients with HL with high risk of residual disease following ASCT ( high risk defined as refractory disease, relapse <12 months, or relapse ≥12 months with extranodal disease after frontline therapy) received nivolumab (240 mg IV every 2 weeks) starting 45-180 days post-transplant for a maximum of 6 months of treatment. Patients were followed for AEs through 100 days after the last dose of drug. PET-CT response assessments were performed 1-3 month, 6 month, and 12 month post-ASCT. The primary objective was to evaluate the safety and tolerability of nivolumab as maintenance therapy early after ASCT. The secondary objective was to evaluate progression-free survival (PFS) at 12 months post-transplant. Results: To date, 37 patients were enrolled; median age 36 years; 25 patients (68%) male. The median number of prior systemic regimens was 2 (range 2-4). 25 patients (68%) had relapsed disease, and 12 patients (32%) had primary refractory disease. 18 patients (49%) had extranodal disease at relapse, 6 patients (16%) had B-symptoms at relapse, and 11 patients (30%) had residual disease after salvage, including 10 patients (27%) of whom had 2-3 prior salvage therapies. 22 patients (60%) had received prior brentuximab, and 3 patients (8%) had received prior nivolumab or pembrolizumab. 36 patients received ASCT and 1 patient received tandem ASCT. At the time of data cutoff, 28 patients (76%) had discontinued nivolumab treatment, 22 patients (60%) because they had completed the 6-month treatment course, 4 patients (11%) due to an adverse event (AE) (1 patient each with pain, pneumonitis, rhabdomyolysis, or hypothyroidism), and 2 patients (5%) due to disease progression. The median duration of treatment was 22.1 weeks. 17 patients (46%) experienced a treatment-related AE (TRAE), of which 5 patients (14%) experienced a ≥Grade 3 TRAE. The most common (≥5%) TRAEs were diarrhea, fatigue, bone pain, neutrophil count decreased, pruritus, rash, and vomiting. 2 patients experienced a treatment-related serious AE (pneumonitis, rhabdomyolysis). There were no treatment-related deaths. With a median follow up of 9.2 months, the median PFS and overall survival (OS) have not been reached. The 6 month PFS is 92.1% and the 12-month OS is 100%. There were no differences in OS when stratified based on prior treatment. Conclusions: The use of nivolumab maintenance early after ASCT is safe and tolerable in this high risk patient population. Early efficacy data is promising, but data need to mature to determine the 12 month PFS. Figure 1 Figure 1. Disclosures Bachier: CRISPR: Membership on an entity's Board of Directors or advisory committees; Autolus: Membership on an entity's Board of Directors or advisory committees; Nkarta: Membership on an entity's Board of Directors or advisory committees; Mana: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Shah: Umoja: Consultancy; Incyte: Consultancy; Legend: Consultancy; Kite: Consultancy; Miltenyi Biotec: Consultancy, Honoraria, Research Funding; Lily: Consultancy, Honoraria, Research Funding; Epizyme: Consultancy.

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