Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death concurrent with down-modulation of phosphoAkt and cyclin D1 and subsequent cellcycle arrest. However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. In the present study, we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes and increased LC3-II and p62 levels. We also show that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagiclysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. This finding provided rationale for examining combination therapy with FTY720 and milatuzumab, an anti- IntroductionMantle cell lymphoma (MCL) is a B-cell malignancy that comprises 3%-8% of non-Hodgkin lymphoma cases diagnosed each year. 1 Whereas the current treatment approach of using combination chemotherapeutic regimens can lead to complete remission, virtually all MCL patients relapse and outcome remains poor, with a median survival of only 3 years. 2 The aggressive clinical behavior of MCL may be because of the complex pathophysiology of the disease, which includes cell-cycle dysregulation driven by cyclin D1 overexpression, alteration in the DNA-damage response, and constitutive activation of key antiapoptotic pathways such as PI3K/Akt and NF-B. [3][4][5][6] Given the absence of curative therapy and the limited number of options for patients with relapsed/refractory MCL, it will be essential to improve our understanding of the complex biology of this disease so that novel treatment approaches can be developed. FTY720 (fingolimod), is a synthetic analog of sphingosine that was developed as an immunosuppressive agent. 7,8 Based on the results of a recent phase 3 clinical trial, FTY720 has been approved by the US Food and Drug Administration (FDA) to treat relapsed multiple sclerosis. 9 We have recently reported that FTY720 has in vitro and in vivo activity in MCL. 10 FTY720 promotes death of MCL cell lines and primary MCL tumor cells via caspaseindependent radical oxygen species (ROS) generation, downmodulation of phospho-Akt and cyclin D1, with accumulation of cells in G 0 /G 1 and G 2 /M phases of the cell cycle. Whereas these data provided information explaining the antitumor activity of FTY720, the effects of this drug on the pathophysiology of MCL required further characterization.In the present study, we show that FTY720 inhibits autophagic flux and induces MCL cell death through lysosomal membrane permeabilization and subsequent translocation of lysosomal hydrolases in the cytosol. Because the autophagy-lysosomal pathway represents an important regulatory mechanism governing the cellular proteome, we hypo...

Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkins lymphoma (NHL) associated with an aggressive clinical course. Inhibition of the ubiquitin-proteasome pathway modulates survival and proliferation signals in MCL and has shown clinical benefit in this disease. This has provided rationale for exploring combination regimens with B-cell selective immunotherapies such as rituximab. In this study, we examined the effects of combined treatment with bortezomib and rituximab on patient-derived MCL cell lines (Jeko, Mino, SP53) and tumor samples from patients with MCL where we validate reversible proteasome inhibition concurrent with cell cycle arrest and additive induction of apoptosis. When MCL cells were exposed to single agent bortezomib or combination bortezomib/rituximab, caspase dependent and independent apoptosis was observed. Single agent bortezomib or rituximab treatment of Mino and Jeko cell lines and patient samples resulted in decreased levels of nuclear NFkappaB complex(es) capable of binding p65 consensus oligonucleotides, and this decrease was enhanced by the combination. Constitutive activation of the Akt pathway was also diminished with bortezomib alone or in combination with rituximab. On the basis of in vitro data demonstrating additive apoptosis and enhanced NFkappaB and phosphorylated Akt depletion in MCL with combination bortezomib plus rituximab, a phase II trial of bortezomib-rituximab in patients with relapsed/refractory MCL is underway.

Aims Porous metaphyseal cones can be used for fixation in revision total knee arthroplasty (rTKA) and complex TKAs. This metaphyseal fixation has led to some surgeons using shorter cemented stems instead of diaphyseal engaging cementless stems with a potential benefit of ease of obtaining proper alignment without being beholden to the diaphysis. The purpose of this study was to evaluate short term clinical and radiographic outcomes of a series of TKA cases performed using 3D-printed metaphyseal cones. Methods A retrospective review of 86 rTKAs and nine complex primary TKAs, with an average age of 63.2 years (SD 8.2) and BMI of 34.0 kg/m2 (SD 8.7), in which metaphyseal cones were used for both femoral and tibial fixation were compared for their knee alignment based on the type of stem used. Overall, 22 knees had cementless stems on both sides, 52 had cemented stems on both sides, and 15 had mixed stems. Postoperative long-standing radiographs were evaluated for coronal and sagittal plane alignment. Adjusted logistic regression models were run to assess malalignment hip-knee-ankle (HKA) alignment beyond ± 3° and sagittal alignment of the tibial and femoral components ± 3° by stem type. Results No patients had a revision of a cone due to aseptic loosening; however, two had revision surgery due to infection. In all, 26 (27%) patients had HKA malalignment; nine (9.5%) patients had sagittal plane malalignment, five (5.6%) of the tibia, and four (10.8%) of the femur. After adjusting for age, sex, and BMI, there was a significantly increased risk for malalignment when a cone was used and both the femur and tibia had cementless compared to cemented stems (odds ratio 3.19, 95% confidence interval 1.01 to 10.05). Conclusion Porous 3D-printed cones provide excellent metaphyseal fixation. However, these central cones make the use of offset couplers difficult and may generate malalignment with cementless stems. We found 3.19-times higher odds of malalignment in our TKAs performed with metaphyseal cones and both femoral and tibial cementless stems. Cite this article: Bone Joint J 2021;103-B(6 Supple A):150–157.

600 Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death via down-modulation of phospho-Akt and Cyclin D1, and subsequent cell cycle arrest (1). However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. Here we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes, increased LC3-II and p62 levels. FTY720 is phosphorylated in vivo by sphingosine kinase 2 and converted to p-FTY720, which binds to sphingosine-1-phosphate (S1P) receptors. A non-phosphorylatable FTY720 derivative (OSU-2S) was recently developed at the Ohio State University (2): OSU-2S treatment induces MCL cell death and shows features of autophagy blockage that led us to conclude that FTY720 phosphorylation and its interaction with SP1 receptors are not required for FTY720-mediated cell death and blockage of autophagy in MCL cells. We also demonstrate that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagic-lysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. We have recently reported CD74 to be expressed on MCL cells and that milatuzumab (Immunomedics, Morris Plains, NJ), a humanized anti-CD74 monoclonal antibody, has significant anti-MCL activity in vitro and in vivo (3). This finding provided the rationale for examining combination therapy with FTY720 and milatuzumab. The in vitro survival of 4 MCL cell lines treated with FTY720, immobilized milatuzumab, and the combination was determined at 24 hours by Annexin-V/PI staining and flow cytometry. Incubation of 4 MCL cell lines with FTY720 and milatuzumab (1 μg/ml) resulted in a statistically significant decrease in cell viability compared to either agent alone for each of the four cell lines (P< 0.01), despite using FTY720 at concentrations lower than the LC50 previously published [Jeko-1 FTY720: 10 μM (LC50: 12.5 μM), Z-138 and UPN-1: 6 μM (LC50: 7.5 μM); Mino 3.75 μM (LC50: 7.5μM)] (1). Notably, combination treatment resulted in synergistic killing in cell lines derived from patients with blastoid variant MCL (Jeko-1, Z-138, UPN-1), despite the fact that both FTY720 and milatuzumab as single agents showed only modest activity. Incubation of primary tumor cells from 6 MCL patients (3 blastoid variant and 3 classic MCL) with FTY720 (2.5 μM, LC50: 5 μM) and miltauzumab induced an average 78.5% cell death compared to 47% of FTY720 treated cells and 50% the milatuzumab-treated cells (P=0.0005 and P=0.0014, respectively). To examine the in vivo activity of FTY720 and milatuzumab, a preclinical model of human MCL using the SCID (CB17 scid/scid) mouse depleted of NK cells was used. In this model, i.v. injection of 40×106 JeKo cells results in disseminated MCL 3 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice (n=10/group) were treated starting at day 15 post engraftment. Twenty control mice received either placebo (saline) or trastuzumab (15 mg/kg) treatment. The third group was treated with FTY720 (5 mg/kg) every day for 2 weeks via i.p injection. The fourth group received milatuzumab (15 mg/kg) every three days, via i.p. injection. The fifth group received the combination of FTY720 and milatuzumab. The median survival for the combination-treated group was 36 days (95% CI:31,36), compared to 28 days for the saline-treated mice (95% CI:24,31), 27 days for the trastuzumab-treated mice (95% CI:23,29), 31 days for the FTY720-treated mice (95% CI:28,32), and 33.5 days for the milatuzumab-treated mice (95% CI:23,34). The combination treatment significantly prolonged survival of this group compared to control groups (P<0.0001), FTY720 (P=0.0001) and milatuzumab (P=0.0048). The most clinically relevant aspect of these findings is that we demonstrate that a potent anti-MCL agent (FTY720) has also the ability to modulate a druggable target (CD74) by preventing its degradation in the autophagic-lysosomal pathway. We believe these findings support clinical evaluation of this combination in patients with MCL. Disclosures: Off Label Use: fty720 immunosupressive drug milatuzumab fully humanized anti-CD74 monoclonal antibody. Goldenberg:Immunomedics: Equity Ownership, Immunomedics owns milatuzumab, Patents & Royalties.

2788 Preclinical studies conducted at our institution (Alinari et al. Blood Abstract 1694, 2009) demonstrated superior efficacy of milatuzumab (Immunomedics, Inc.), a humanized anti-CD74 antibody, in combination with rituximab in mantle cell lymphoma (MCL) cell lines, MCL patient (pt) primary tumor cells, and an in vivo preclinical model of human MCL, compared to either agent alone. Veltuzumab (Immunomedics, Inc.), a novel humanized anti-CD20 antibody, has been reported to have several advantages over rituximab including slower off-rates, shorter infusion times, higher potency, and improved therapeutic responses in animal models. Phase II clinical testing of veltuzumab demonstrated single agent activity in pts with relapsed and refractory non-Hodgkin's lymphoma (NHL). In vitro, veltuzumab combined with milatuzumab also resulted in enhanced apoptosis compared to either agent alone, similar to that observed with rituximab and milatuzumab (Fig 1). As a result of the anti-tumor activity observed in vitro with combined veltuzumab and milatuzumab, we initiated a phase I/II trial in pts with relapsed or refractory B-cell NHL after at least 1 prior therapy to determine the safety, tolerability, and overall response rate with this combination. Patients received veltuzumab 200 at mg/m2 weekly combined with escalating doses of milatuzumab at 8, 16, and 20 mg/kg twice per wk of wks 1–4, 12, 20, 28, and 36. All pts received pre-medication with acetominophen, diphenhydramine, and famotidine prior to each veltuzumab dose and acetominophen, diphenhydramine, and hydrocortisone 50 mg before and after each milatuzumab dose. Dose limiting toxicity was defined during weeks 1–4. Six pts with grade 2 (n=2) or 3 (n=4) follicular NHL, have completed at least 4 weeks of combined veltuzumab and milatuzumab. Median age was 63.5 years (range 44–81), and pts received a median of 3.5 prior therapies (range 3 – 5), including 2 pts with prior autologous stem cell transplant. Three of 6 patients were refractory to rituximab, defined as having less than a partial response to the last rituximab-containing regimen. Dose escalation has reached 16 mg/kg milatuzumab, and no dose limiting toxicities have been observed to date. However, 3 of 6 pts experienced grade 3 infusion reactions during or immediately following the milatuzumab infusion; 1 pt treated with 8 mg/kg milatuzumab during weeks 1 and 12, and 2 pts receiving 16 mg/kg during weeks 3 and 12. Grade 3 infusion reactions consisted of fever, rigors, nausea, vomiting, diarrhea, and in 1 case a diffuse macular rash. Grade 3–4 hematologic toxicity occurred in only 2 pts consisting of grade 4 lymphopenia, and no infections have been observed. The most frequently observed grade 1–2 toxicities were fatigue, transient hyperglycemia, dyspnea, hypoalbuminemia, and thrombocytopenia. Human anti-veltuzumab and anti-milatuzumab antibodies, collected in all 6 pts pretreatment and day 1 of weeks 4 and 12, have not been detected in any pt. Plasma cytokine levels of IL-10, IL-12, TNF-α and INF-γ were checked pre- and post- veltuzumab infusion on day 1 and pre- and post-milatuzumab infusion on day 2. While elevations in cytokine levels were observed, there was no correlation with infusion reactions or response. In the first cohort, one pt achieved a PR maintained at week 20, 1 pt experienced stable disease at week 10, and 1 pt developed progressive disease at week 20. In the second cohort, two pts achieved a PR at week 10, and 1 pt had stable disease at week 10. Two of 3 responding pts were rituximab-refractory. Due to the observed infusion reactions with milatuzumab, the protocol has been amended to include additional premedication with intravenous antihistamine, and dexamethasone 20 mg pre-milatuzumab and 10 mg post-milatuzumab. The schedule of treatment was also modified so that the antibodies are no longer administered on the same day. Dose escalation will continue and updated results will be presented. The first 2 cohorts will be expanded to 6 pts to determine if the modifications will limit the infusion reactions and permit prolonged dosing in responding pts. In summary, 3 of 6 pts with relapsed or refractory NHL treated to date including heavily pretreated and rituximab-refractory pts, responded to combination therapy, achieving a PR. The primary observed toxicity has been infusion reactions due to milatuzumab. Disclosures: Christian: Immunomedics, Inc.: Research Funding. Off Label Use: The use of the monoclonal antibodies veltuzumab and milatuzumab is experimental in the treatment of non-Hodgkin's lymphoma. Benson:Immunomedics, Inc: Research Funding. Jones:Immunomedics, Inc.: Research Funding. Wegener:Immunomedics, Inc.: Employment, shareholders. Goldenberg:Immunomedics, Inc.: Employment, shareholders. Baiocchi:Immunomedics, Inc.: Research Funding. Blum:Immunomedics, Inc.: Research Funding.