•This is the first prospective trial in the US comparing tbo-filgrastim and filgrastim for stem cell mobilization in patients undergoing autologous stem cell transplantation.•Tbo-filgrastim is not ...inferior to filgrastim in stem cell yield on the first day of apheresis.•A trend toward greater mobilization was noted in tbo-filgrastim arm.•No differences in safety, engraftment outcomes, and hospital readmission rates were noted between the study arms.
Autologous hematopoietic stem cell transplantation (auto-HSCT) improves survival in patients with multiple myeloma (MM) and non-Hodgkin lymphoma (NHL). Traditionally, filgrastim (Neupogen; recombinant G-CSF) has been used in as a single agent or in combination with plerixafor for stem cell mobilization for auto-HSCT. In Europe, a biosimilar recombinant G-CSF (Tevagrastim) has been approved for various indications similar to those of reference filgrastim, including stem cell mobilization for auto-HSCT; however, in the United States, tbo-filgrastim (Granix) is registered under the original biological application and is not approved for stem cell mobilization. In retrospective studies, stem cell mobilization with tbo-filgrastim has shown similar efficacy and toxicity as filgrastim, but no prospective studies have been published to date. We have conducted the first prospective randomized trial comparing the safety and efficacy of tbo-filgrastim in combination with plerixafor with that of filgrastim in combination with plerixafor for stem cell mobilization in patients with MM and NHL. This is a phase 2 prospective randomized (1:1) open-label single-institution noninferiority study of tbo-filgrastim and filgrastim with plerixafor in patients with MM or NHL undergoing auto-HSCT. Here 10 µg/kg/day of tbo-filgrastim/filgrastim was administered s.c. for 5 days (days 1 to 5). On day 4 at approximately 1800 hours, 0.24 mg/kg of plerixafor was administered s.c. Apheresis was performed on day 5 with a target cumulative collection goal of at least 5.0 × 106 CD34+ cells/kg. The primary objective was to compare day 5 CD34+ cells/kg collected. Secondary objectives included other mobilization endpoints, safety, engraftment outcomes, and hospital readmission rate. A total of 97 evaluable patients were enrolled (tbo-filgrastim, n = 46; filgrastim, n = 51). Tbo-filgrastim was not inferior to filgrastim in terms of day 5 CD34+ cell collection (mean, 11.6 ± 6.7 CD34+ cells/kg versus 10.0 ± 6.8 CD34+ cells/kg. Multivariate analysis revealed a trend toward increased mobilization in the tbo-filgrastim arm, but this was not statistically significant. The tbo-filgrastim and filgrastim arms were similar in all secondary endpoints. Tbo-filgrastim is not inferior in efficacy and has similar safety compared to reference filgrastim when used for stem cell mobilization in patients with MM and NHL. Granix can be safely used instead of Neupogen for stem cell collection in patients undergoing auto-HSCT for MM or NHL.
The study is registered at https://clinicaltrials.gov/ct2/show/NCT02098109.
Introduction: Allogeneic hematopoietic cell transplantation is a cornerstone of therapy for hematologic malignancies and often a patient's only curative intent treatment. Traditionally reserved for ...patients with an HLA-matched donor, advances in graft versus host disease (GVHD) prophylaxis utilizing post-transplant cyclophosphamide (PTCy) have expanded the use of haploidentical hematopoietic cell transplantation (haplo-HCT). While overall outcomes for haplo-HCT appear to be excellent, its novel approach brings toxicities that are particular to its biological and clinical milieu.
The immediate post-transplant course in T cell replete haplo-HCT is often complicated by symptoms including fever, hypoxia, hypotension and organ dysfunction resembling the cytokine release syndrome (CRS) previously described in recipients of targeted cellular therapeutics such as CAR T-Cells. IL-6 is thought to be a key mediator of CRS in patients receiving novel T-cell engaging therapies, and tocilizumab has been used with success in this setting.
Here we aim to describe the nature and incidence of CRS in haplo-HCT recipients, as well as its potential implications on clinical outcomes. Additionally, prospective analysis of cytokine profiles of haplo-HCT recipients and clinical responses to tocilizumab therapy are presented.
Patients and Methods: We performed a retrospective review of patients who underwent haplo-HCT transplantation at our institution from July 2009 through April 2015. Patients were scored for symptoms of CRS based on established grading criteria (Lee et al, Blood 2014). Patients were stratified into three categories by grade of CRS experienced: none (grade 0), mild (grade 1-2) and severe (grade 3-4). Outcomes were assessed.
A total of 84 patients were identified, 55% (46) were male, with a median age at transplant of 49 (19-73), and 49% (41) had active disease at the time of transplant. The most common diagnosis was AML (55 pts), followed by ALL (9 pts), MDS (5 pts) and NHL (2 pts). Among the patients, 26% (22 pts) had undergone prior transplant.
In addition to the retrospective review, baseline and post- transplant cytokine levels were prospectively drawn in 10 patients who underwent haplo-HCT. A total of 7 additional patients who met criteria for CRS were treated prospectively with tocilizumab (dose: 4mg/kg-bw) and clinical responses were recorded. We recorded CRP levels in selected patients as part of clinical monitoring.
Results: We found a high incidence, 85%, of CRS in our haplo-HCT patients. Among a total of 84 patients, 12 (14%) experienced severe CRS, 60 (72%) had mild CRS, and 12 (14%) patients had no evidence of CRS. The most common manifestations of severe CRS included: fever (100%), respiratory failure (75%), hypotension (83.3%), hepatic failure (25%) and renal failure (33.3%). The median maximum CRP (during post-transplant days 0 to 8) in all patients suffering from CRS was 155 mg/L.
Of the twelve patients who suffered from severe CRS, nine (75%) died. Predicted median survival was 0.72 months for severe CRS, 12.7 months for patients with no CRS and was not reached for patients with mild CRS (fig 1). Rates of acute and chronic graft-versus-host disease did not differ by CRS status. The incidence of mild and severe CRS did not differ by ABO mismatch, age, CMV status, donor sex, T-cell or CD34 cell dose. There was no difference in rates of CRS for patients in remission versus active disease at time of transplant. There were no differences in engraftment.
Cytokine profiles in haplo-HCT recipients showed significant elevation in serum IL-6 levels, most significant in patients who suffered from severe CRS (fig 2). We administered tocilizumab to 7 patients with severe CRS symptoms early after haplo-HCT (median day of treatment was day +3) resulting in the resolution of their CRS symptoms within 48-72 hours. Over the same time period CRP levels dropped below 50% of the peak value.
Summary:
CRS is common after T-cell replete haplo-HCT and severe CRS is potentially associated with high risk of early mortality after transplant. Cytokine profiles suggest IL-6 is a key mediator of CRS in haplo-HCT patients. Tocilizumab appears to be an effective treatment for patients who develop severe CRS early after T-cell replete haplo-HCT. Future prospective studies are warranted in studying the role of tocilizumab in the treatment and potentially prevention of severe CRS in haplo-HCT patients.
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Fehniger:Celgene: Research Funding. Uy:Novartis: Research Funding. Abboud:Teva Pharmaceuticals: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Pfizer: Research Funding; Merck: Research Funding; Gerson Lehman Group: Consultancy.
Background: Multiple myeloma (MM) is a hematologic cancer characterized by a diversity of genetic lesions-translocations, copy number alterations (CNAs), and single nucleotide variants (SNVs). The ...prognostic value of translocations and of CNAs has been well established. Determining the clinical significance of SNVs, which are recurrently mutated at much lower frequencies, and how this significance is impacted by translocations and CNAs requires additional, large-scale correlative studies. Such studies can be facilitated by cost-effective targeted sequencing approaches. Hence, we designed a single-platform targeted sequencing approach capable of detecting all three variant types.
Methods: We designed oligonucleotide probes complementary to the coding regions of 467 genes and to the IgH and MYC loci, allowing a probe to closely match at most 5 regions within the genome. Genes were selected if they were expressed in an independent RNA-seq MM data set and harbored germline SNP-filtered variants that: (1) occurred with frequency >3%, (2) were clustered in hotspots, (3) occurred in recurrently mutated "cancer genes" (as annotated in COSMIC or MutSig), or (4) occurred in genes involved in DNA repair and/or B-cell biology. IgH and MYC tiling was unbiased (with respect to annotated features within the loci) and spanned from 50 kilobasepairs (kbps) upstream of both regions to 50 kbps downstream of IgH and 100 kbps downstream of MYC.
Results: We performed targeted sequencing of 96 CD138-enriched samples derived from MM patients, as well as matched peripheral blood leukocyte normal controls. Sequencing depth (mean 107X) was commensurate with that of available exome sequencing data from these samples (mean 71X). Samples harbored a mean of 25 non-silent variants, including those in known MM-associated genes: NRAS (24%), KRAS (22%), FAM46C (17%), TP53 (10%), DIS3 (8%), and BRAF (3%). Variants detected by both platforms showed a strong correlation (r^2 = 0.8). The capture array detected activating, oncogenic variants in NRAS Q61K (n=3 patients) and KRAS G12C/D/R/V (n=5) that were not detected in exome data. Additionally, we found non-silent, capture-specific variants in MTOR (3%) and in two transcription-related genes that have been previously implicated in cancer: ZFHX4 (5%) and CHD3 (5%). To assess the potential role of deep subclonal variants and our ability to detect them, we performed additional sequencing (mean 565X) on six of the tumor/normal pairs. This revealed 14 manually-reviewed, non-silent variants that were not detected by the initial targeted sequencing. These had a mean variant allele frequency of 2.8% and included mutations in DNMT3A and FAM46C. At least one of these 14 variants occurred in five of the six re-sequenced samples. This highlights the importance of this additional depth, which will be used in future studies. Our approach successfully detected CNAs near expected frequencies, including hyperdiploidy (52%), del(13) (43%), and gain of 1q (35%). Similarly, it inferred IgH translocations at expected frequencies: t(4;14) (14%), t(6;14) (3%), t(11;14) (15%), and t(14;20) (1%). As expected, translocations occur predominantly within the IgH constant region, but also frequently 5' (i.e., telomeric) of the IGHM switch region, and occasionally within the V and D regions. We detected MYC -associated translocations, whose frequencies have been the subject of debate, at 10% (n=9 patients), with five involving IgH, three having both partners in or near MYC, and one having both types. Finally, our platform detected novel IgH translocations with partners near DERL3 (n=2), MYCN (n=1), and FLT3 (n=1). Additional evidence suggests that DERL3 and MYCN may be targets of IgH-induced overexpression: of 84 RNA-seq patient samples, six exhibited outlying expression of DERL3, including one sample in which we detected the translocation in corresponding DNA, and one exhibited outlying expression of MYCN.
Conclusion: Our MM-specific targeted sequencing strategy is capable of detecting deeply subclonal SNVs, in addition to CNAs and IgH and MYC translocations. Though additional validation is required, particularly with respect to translocation detection, we anticipate that such technology will soon enable clinical testing on a single sequencing platform.
Vij:Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy; Takeda, Onyx: Research Funding.
Abstract 2261
Plerixafor (AMD3100) is a CXCR4 antagonist that was approved by the FDA for use in combination with G-CSF to mobilize hematopoietic stem and progenitor cells (HSPCs) in patients with ...non-Hodgkin's lymphoma and multiple myeloma. We have previously reported the use of plerixafor alone to mobilize HSPCs for HLA-matched allogeneic transplantation (Blood. 2008;112:990). In this trial, sibling donors were treated with plerixafor at a dose of 0.24 mg/kg by subcutaneous (SC) injection, followed 4 hours later by leukapheresis. To date, 15 of 45 donors (33%) required a second day of leukapheresis to collect the minimum cell dose of ≥ 2.0 × 106 CD34+ cells/kg actual recipient body weight that are required to proceed to transplantation. Based on preliminary data suggesting higher (2-fold) and earlier (1 hr vs. 3 hr) progenitor mobilization in mice after intravenous (IV) dosing of plerixafor, we amended our trial to test the safety and efficacy of IV plerixafor.
In our Phase I trial, 21 healthy donors were initially mobilized with increasing doses of IV plerixafor (0.08, 0.16, 0.24, 0.32, 0.40 or 0.48 mg/kg). After 4 days of drug clearance, the same donors were then mobilized with a single SC dose of 0.24 mg/kg plerixafor followed 4 hours later by leukapheresis. In our Phase II study, 28 sibling donors were treated with plerixafor at a dose of 0.32 mg/kg by IV injection, followed 4 hours later by leukapheresis. Successful mobilization was defined as a minimum leukapheresis yield of ≥ 2.0 × 106/kg CD34+ cell/kg actual recipient body weight.
Peak CD34+ cells/mm3 were observed 4 hours after IV dosing (vs. 6–9 hours after SC dosing) and donors given 0.24 mg/kg IV plerixafor, had significantly higher peak levels of CD34+ cells/mm3 compared to the same donors who received 0.24 mg/kg SC plerixafor. There was a clear dose-response relationship of IV plerixafor on mobilization of CD34+ HSPCs, with the 0.32 mg/kg dose yielding a maximum increase in circulating CD34+ cells of 27 CD34+ cells/mm3 at 4 hours after injection, representing a median eight-fold increase from baseline. Interestingly, the median CD34+ cells/mm3 in donors receiving 0.40 mg/kg (N=3) and 0.48 mg/kg (N=3) was 17 and 24 CD34+ cells/mm3, respectively, not significantly better than the 320 mcg/kg dose at the four hour time-point. We also noted that IV dosing (especially doses >0.24 mg/kg) resulted in prolonged mobilization of CD34+ cells such that levels approached 20 CD34+ cells/mm3 at 24 hours after IV dosing. Pharmacokinetic studies demonstrated that the Cmax of plerixafor following the 0.32 mg/kg IV dose remained below 1.0 μg/mL whereas the 0.40 and 0.48 mg/kg doses resulted in Cmax levels of 1.8–2.2 μg/mL. A total of 28 HLA-identical sibling donor/recipient pairs have been enrolled in the Phase II trial, with all donors mobilized using IV plerixafor at a dose 0.32 mg/kg. At 4 hours after IV plerixafor, the CD34+ cell count rose to a median of 16 CD34+ cells/mm3 (range, 4–46), representing a 6.5-fold increase. The efficacy of CD34+ cell collection following 0.32 mg/kg IV plerixafor was not evaluable in five donors due to incomplete apheresis collections or the extremely large size of one recipient (recipient >200 kg). Six of the remaining 23 donors (26%) did not achieve the minimum cell dose of 2 × 106 CD34+ cells/kg in a single 20 L leukapheresis procedure. This mobilization failure rate of 26% with 0.32 mg/kg IV plerixafor is similar to the failure rate of 33% we observed following administration of 0.24 mg/kg SC plerixafor to healthy donors. Four of the six patients who failed to collect ≥ 2.0 × 106 CD34+ cells/kg after the first mobilization and apheresis procedure reached goal following a second mobilization and collection procedure. No adverse events or acute grade 3 or 4 toxicities have been observed in any of the donors given IV plerixafor doses up to 0.48 mg/kg. We have transplanted 24 of the 28 patients entered in the phase II trial. All but one patient engrafted neutrophils and platelets promptly and only 2 of the 24 (8.3%) transplant recipients have developed clinical evidence of acute GVHD (minimum follow-up of 45 days); 1 had grade II and the other grade III.
IV plerixafor increases the magnitude and duration of CD34+ mobilization compared to SC plerixafor in healthy donors. These observations suggest that IV plerixafor may be a more effective mobilization agent with a low side effect profile for allogeneic transplantation.
Rettig: Genzyme Corp.: Consultancy, Honoraria. DiPersio: Genzyme: Honoraria.
Background: MDS is a genetically complex, oligoclonal disease consisting of a founding clone and typically one or more subclones derived from the founding clone. Previously we have shown that in MDS ...patients treated with chemotherapy, a minor subclone present at diagnosis can expand during disease progression from MDS to secondary AML, highlighting the clinical implications of clonal heterogeneity. Whether a similar pattern of clonal evolution occurs in MDS patients that relapse following allogeneic hematopoietic stem cell transplant (alloHSCT) is not known.
Methods: We identified 9 MDS patients who progressed after receiving either a myeloablative (n=3) or reduced-intensity (n=6) alloHSCT (median time to progression 309 days, range 98-881). We performed enhanced exome sequencing (EES) to define the clonal architecture of 23 tumor samples at the following clinical landmarks (with matched skin as a source of normal DNA): diagnosis, <2 months pre-alloHSCT (where available), and relapse post-alloHSCT. Somatic mutations were validated in the 23 discovery samples and genotyped in 35 additional serial banked samples at various time-points post-alloHSCT, including day 30 and 100, using capture probes targeting all putative single-nucleotide variants (SNV) and short insertions and deletions (INDELs) identified by EES. The variant allele fraction (VAF) of all validated somatic mutations was determined. Ultra-deep, error-corrected sequencing (i.e., barcoded sequencing) was performed on 49 tumor samples to provide increased sensitivity to detect low-level mutations post-alloHSCT. Copy number alterations were identified using exome and SNP array data.
Results: Averaged sequencing coverage depth was 246x for tumors subjected to EES; 537x for validation sequencing, and 24,150x total and 5,180x unique for the ultra-deep sequencing. In all cases, we observed that mutations found in the diagnostic founding clone were always detected at relapse. However, using SNVs, INDELs, and copy number analysis, we show that the dominant clone at relapse was often derived from a population that was subclonal at presentation. We observed the following, non-mutually exclusive patterns of clonal evolution at relapse: i) A subclone expanded or emerged and became the dominant clone at relapse as compared to presentation (n=6). In 2 of these cases, the subclone contained mutations that were not detected at presentation even via ultra-deep sequencing. ii) A subclone was cleared with alloHSCT (defined as VAF<1% by EES, n=4), confirmed by ultra-deep sequencing when available (n=2). iii) The founding/dominant clone at diagnosis was also the dominant clone at relapse (n=3). However, in 2 of these 3 cases, changes in clonal architecture were observed with evidence of rising or contracting subclones. Although our sample size is relatively small, the intensity of the alloHSCT conditioning regimen did not impact the relapse pattern. No acquired abnormalities were detected in the MHC locus, and no mutations in a particular gene family or cellular pathway were consistently observed in rising or contracting subclones.
Finally, we used ultra-deep sequencing to determine if mutations (i.e., tumor cells) could be detected at day 30 post-alloHSCT in 7 of the 8 patients with no evidence of disease, who had available data. Mutations were detected in 6 of 7 patients. The average detectable mutation VAF per patient was 0.37% (ranged from 0.04% to 0.93%)(i.e., 1 mutant cell in 135).
Conclusion: Complex clonal dynamics and clonal evolution are observed at relapse post-alloHSCT for MDS. Although minor subclones rise and may become the dominant clone at relapse, mutations present in the dominant (i.e., founding) clone of the diagnostic MDS sample pre-alloHSCT are always detected at relapse. This is similar to the pattern of clonal evolution previously observed for MDS progression to secondary AML following chemotherapy. These observations have implications for targeted therapy and tumor burden monitoring. Ultra-deep sequencing can detect persistent or emerging mutations at early time-points post-alloHSCT that are associated with subsequent relapse. The predictive value of detecting persistent mutations early after post-alloHSCT merits testing in future studies.
Jacoby:Quintiles: Consultancy; Sunesis: Research Funding; Celgene: Speakers Bureau. DiPersio:Incyte Corporation: Research Funding.
Background: Haploidentical HSCT is a readily available platform for most patients lacking an HLA-matched donor, but the T-cell depleted (TCD) approach without any post-transplant donor cell therapy ...is impaired by high non-relapse mortality (NRM) due to slow IR, while the T-cell replete (TCR) approach followed by cyclophosphamide and immunosuppressive therapy is hampered by high chronic GvHD and relapse incidence (RI)
Methods: The impactof IR and TK-cell dose on outcomes was assessed in 45 patients with several hematological malignancies treated with 1 to 4 monthly TK infusions (0.1-1.0x107/Kg, 21-49 days after haploidentical HSCT) in a phase 2 trial (n=30; Lancet Oncol 2009; 10: 489) and in the experimental arm of an ongoing phase 3 trial (n=15; NCT00914628). Median follow-up time for TK-treated patients was 3.7 years (interquartile range IQR, 1.5-8.5). Outcome measures included 1-year NRM, overall survival (OS), leukemia-free survival (LFS), RI and GvHD. Hazard ratios (HR) and odds ratios (OR) were derived by regression models adjusted for baseline risk factors. IR (T cells ≥ 100/µL) was modelled as a time-dependent covariate. These pooled data were the basis for the positive opinion for conditional marketing authorization recently granted for TK cells (www.ema.europe.eu/EPAR/Zalmoxis)
Results: IR was achieved by 34 patients (76%) after a median of two TK infusions (IQR, 1-2), a median cumulative cell dose of 1.3x107/kg (1.0-2.4) and a median time from HSCT of 83 days (65-108). IR was not influenced by baseline risk factors or TK-cell doses and was associated with decreased NRM (12% vs 75%; p<0.0001) and improved LFS (HR=0.20; p=0.005) and OS (HR=0.08; p<0.0001). Grade II-IV acute GvHD (35%; grade III-IV: 7%) was unrelated to TK-cell dose and occurred nearly exclusively in IR (OR=7.0; p=0.07) and female patients (OR=9.7; p=0.007). Only one patient had de novo chronic GvHD. All GvHD events fully resolved (median, 14 days; 10-27) after suicide-gene induction with ganciclovir (median, 15 days; 13-16). Cumulative RI (31%) did not differ according to IR, while it was inversely related to TK-cell dose, with decreased relapse rates (60%, 33% and 0%) observed with increasing cell doses (<1.0, 1.0-2.4 and >2.4x107, respectively; p=0.004). TK patients had significant outcomes in OS (51%), NRM (20%) and chronic GvHD (6%). Importantly, TK patients with grade II-IV acute GvHD treated with ganciclovir experienced high OS rates (67%), thus confirming the ability of TK machinery to selectively targeting GvHD effector cells, while sparing a wide repertoire protective against infections and leukemia recurrence
Conclusions: TK-cell treatment is characterized by early IR and full GvHD control that translate in improved NRM and OS and by dose-related antileukemic effects, with overall outcomes comparing very favorably with those of current approaches to haploidentical HSCT
Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Colombi:MolMed: Employment. Lambiase:MolMed: Employment. Bordignon:MolMed SpA: Employment.
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