Minimal Residual Disease State Research Articles

Fludarabine and rituximab with escalating doses of lenalidomide followed by lenalidomide/rituximab maintenance in previously untreated chronic lymphocytic leukaemia (CLL): the REVLIRIT CLL-5 AGMT phase I/II study

Despite recent advances, chemoimmunotherapy remains a standard for fit previously untreated chronic lymphocytic leukaemia patients. Lenalidomide had activity in early monotherapy trials, but tumour lysis and flare proved major obstacles in its development. We combined lenalidomide in increasing doses with six cycles of fludarabine and rituximab (FR), followed by lenalidomide/rituximab maintenance. In 45 chemo-naive patients, included in this trial, individual tolerability of the combination was highly divergent and no systematic toxicity determining a maximum tolerated dose was found. Grade 3/4 neutropenia (71%) was high, but only 7% experienced grade 3 infections. No tumour lysis or flare > grade 2 was observed, but skin toxicity proved dose-limiting in nine patients (20%). Overall and complete response rates after induction were 89 and 44% by intention-to-treat, respectively. At a median follow-up of 78.7 months, median progression-free survival (PFS) was 60.3 months. Minimal residual disease and immunoglobulin variable region heavy chain mutation state predicted PFS and TP53 mutation most strongly predicted OS. Baseline clinical factors did not predict tolerance to the immunomodulatory drug lenalidomide, but pretreatment immunophenotypes of T cells showed exhausted memory CD4 cells to predict early dose-limiting non-haematologic events. Overall, combining lenalidomide with FR was feasible and effective, but individual changes in the immune system seemed associated with limiting side effects. clinicaltrials.gov (NCT00738829) and EU Clinical Trials Register (www.clinicaltrialsregister.eu, 2008-001430-27)

Advances in immunotherapy for acute myeloid leukemia.

Evasion of the host immune system is a key mechanism to promote malignant progression. Therapeutically targeting immune pathways has radically changed the treatment paradigm for solid and lymphoid tumors but has yet to be approved for myeloid malignancies. Here, we summarize the most recent advances in immunotherapy for acute myeloid leukemia. Topics reviewed here include adoptive cellular approaches (chimeric antigen receptor-T cells, natural killer and other immune cells), checkpoint inhibitors (anti-PD-1/PD-L1, anti-CTLA-4 and TIM-3) and vaccines (WT-1, HLA-A2 and hTERT). Emphasis is placed on agents with clear evidence of tumor-specific immune responses and/or clinical activity in early-phase trials. Despite concerns regarding heterogeneous antigen expression and cytokine release syndrome, immunotherapy remains a highly promising strategy for acute myeloid leukemia, particularly transplant-ineligible patients and minimal residual disease states.

Is molecular remission the goal of multiple myeloma therapy?

The increased number of effective therapies and the wider use of combinations that give deeper remissions have resulted in a reassessment of the goals of myeloma therapy. With the advent of new therapeutic strategies and diagnostic tools, achievement of minimal residual disease (MRD)-negative status has become increasingly important, with some even considering it as the primary endpoint for therapy. The level of MRD that is aimed for is a continuous, rather than an absolute variable, with studies in both transplant-eligible and -noneligible patients showing that the level of MRD achieved is predictive of progression-free survival and overall survival, with an improvement in survival of approximately 1 year for each log-depletion in MRD level. The most widely used methods to assess MRD status include flow cytometry and clonality detection, using next-generation sequencing technologies with sensitivity limits of 1:10-3 to 1:10-6 The timing of when to assess MRD depends on the treatment used, as well as the molecular and cytogenetic subgroup of the myeloma itself. It is also becoming clear that the level of MRD negativity, as well as microenvironmental factors, are important prognostically, including the regeneration of normal plasma cells, and the normalization of the immune repertoire. With advances in antibody-based therapy and immunotherapy, the achievement of stable MRD states is now possible for a significant proportion of patients, and is a prerequisite for myeloma cure.

Lenalidomide maintenance after first-line therapy for high-risk chronic lymphocytic leukaemia (CLLM1): final results from a randomised, double-blind, phase 3 study

The combined use of genetic markers and detectable minimal residual disease identifies patients with chronic lymphocytic leukaemia with poor outcome after first-line chemoimmunotherapy. We aimed to assess lenalidomide maintenance therapy in these high-risk patients. In this randomised, double-blind, phase 3 study (CLLM1; CLL Maintenance 1 of the German CLL Study Group), patients older than 18 years and diagnosed with immunophenotypically confirmed chronic lymphocytic leukaemia with active disease, who responded to chemoimmunotherapy 2-5 months after completion of first-line therapy and who were assessed as having a high risk for an early progression with at least a partial response after four or more cycles of first-line chemoimmunotherapy, were eligible if they had high minimal residual disease levels or intermediate levels combined with an unmutated IGHV gene status or TP53 alterations. Patients were randomly assigned (2:1) to receive either lenalidomide (5 mg) or placebo. Randomisation was done with a fixed block size of three, and was stratified according to the minimal residual disease level achieved after first-line therapy. Maintenance was started with 5 mg daily, and was escalated to the target dose of 15 mg. If tolerated, medication was administered until disease progression. The primary endpoint was progression-free survival according to an independent review. The pre-planned interim analysis done by intention to treat was done after 20% of the calculated progression-free survival events. This study is registered with ClinicalTrials.gov, number NCT01556776; treatment in the lenalidomide group is still ongoing. Between July 5, 2012, and March 15, 2016, 468 previously untreated patients with chronic lymphocytic leukaemia were screened for the study; 379 (81%) were not eligible. Recruitment was closed prematurely due to poor accrual after 89 of 200 planned patients were randomly assigned: 60 (67%) enrolled patients were assigned to the lenalidomide group and 29 (33%) to the placebo group, of whom 56 (63%) received lenalidomide and 29 (33%) placebo, with a median of 11·0 (IQR 4·5-20·5) treatment cycles at data cutoff. After a median observation time of 17·9 months (IQR 9·1-28·1), the hazard ratio for progression-free survival assessed by an independent review was 0·168 (95% CI 0·074-0·379). Median progression-free survival was 13·3 months (95% CI 9·9-19·7) in the placebo group and not reached (95% CI 32·3-not evaluable) in the lenalidomide group. The most frequent adverse events were skin disorders (35 patients [63%] in the lenalidomide group vs eight patients [28%] in the placebo group), gastrointestinal disorders (34 [61%] vs eight [28%]), infections (30 [54%] vs 19 [66%]), haematological toxicity (28 [50%] vs five [17%]), and general disorders (28 [50%] vs nine [31%]). One fatal adverse event was reported in each of the treatment groups (one [2%] patient with fatal acute lymphocytic leukaemia in the lenalidomide group and one patient (3%) with fatal multifocal leukoencephalopathy in the placebo group). Lenalidomide is an efficacious maintenance therapy reducing the relative risk of progression in first-line patients with chronic lymphocytic leukaemia who do not achieve minimal residual disease negative disease state following chemoimmunotherapy approaches. The toxicity seems to be acceptable considering the poor prognosis of the eligible patients. The trial independently confirms the clinical significance of a novel, minimal residual disease-based algorithm to predict short progression-free survival, which might be incorporated in future clinical trials to identify candidates for additional maintenance treatment. Celgene Corporation.

The evolving role of chemotherapy and hematopoietic cell transplants in Ph-positive acute lymphoblastic leukemia in adults.

The introduction of the tyrosine kinase inhibitors (TKI) into the treatment of patients with Ph or BCR-ABL1-positive acute lymphoblastic leukemia has revolutionized the treatment of this poor prognosis acute leukemia. The combination of TKI with chemotherapy has improved response rates and allowed more patients to proceed to allogeneic hematopoietic cell transplant (alloHCT). Older patients have excellent responses to TKI and corticosteroids or in combination with minimal chemotherapy. This raises the question as to whether patients require full-intensity chemotherapy with TKI to achieve molecular remissions. The pediatricians have proposed that cure is achievable without alloHCT in children. These results have suggested that many patients may not require traditional chemotherapy in addition to TKI to achieve remission, and that patients who achieve a negative minimal residual disease state may not require alloHCT. The data in support of these questions is presented here and a suggested future clinical trial design based on these data is proposed.

Achievement of a negative minimal residual disease state after hypomethylating agent therapy in older patients with AML to reduce risk of relapse.

7018 Background: Persistence of minimal residual disease (MRD) post therapy is a powerful predictor of outcome in patients with AML treated with traditional cytarabine and anthracycline based regimens. The clinical relevance of MRD in the context of hypomethylating agents has not been evaluated extensively. Methods: Among 194 patients with AML treated with single agent azacytidine, decitabine, or gaudecitabine, 116 (median age 76, range 60 - 92) had MRD analysis performed on bone marrow specimens obtained at time of assessment of response or thereafter; among them 69 (59%) achieved either morphologic complete remission (CR) or CR with incomplete recovery of platelets (CRp) or counts (CRi), and 61 (53%) had evaluable MRD data; MRD was assessed using an 8-color flow panel, with a detection sensitivity of 0.01%. Results: Median cycles to achieving response was 2 (range, 1-6). Sixty one patients had evaluable MRD data at the time of response, of whom 19 (28%) became MRD negative (-). This was associated with a reduced cumulative risk of relapse (p = 0.012) but did not translate to an improved relapse-free survival (RFS; p = 0.17) or overall survival (OS; p = 0.79) due to high frequency of non-relapse deaths (attributable to comorbidities and infections) in the MRD- group. Patients who achieved a MRD- state at the time of achieving response had a higher mortality [5/8 (62%)] when compared with those who achieved a MRD- state in subsequent cycles [1/13 (7.6%); p = 0.01], resulting in an inferior OS (6.2 months (mo) vs 20 mo, p = 0.012). Similarly, achieving negative MRD at CR and at any time up to 3 months post response was not associated with improved RFS or OS despite a lower cumulative risk of relapse (p = 0.045). There was no impact of MRD, on OS, whether the MRD- state was achieved after 1st, 3rd or 6thcycle of therapy. Association between depth of MRD response at time of remission and RFS was borderline significant (p = 0.08). On multivariate analysis, response (CR vs CRi/CRp), but not a negative MRD, was predictive for RFS or OS. Conclusions: In this cohort of older AML patients treated with hypomethylating agents, achieving a MRD- state was associated with a reduced risk of relapse but not improved RFS or OS.

Detection of minimal residual disease in childhood B-acute lymphoblastic leukemia by 4-color flowcytometry

Monitoring of minimal residual disease (MRD) is currently considered the most powerful predictor of outcome in acute lymphoblastic leukemia (ALL). Achievement of a negative MRD state assessed by multicolor flowcytometry (MFC) is an important predictor of disease-free survival (DFS) and overall survival (OS) in ALL patients. We sought to determine whether panels of antibodies combination are more suitable for detection of MRD in Childhood ALL. Eighty-four (84) patients with ALL (B-lineage subtype) were enrolled in this study. Normal template for B cell precursors was established in 15 controlparticipants using 4-four panels of monoclonal Antibodies (Mo Abs),{CD22, CD45, CD58 and CD97 in combination with CD10, CD19, CD34}. At diagnosis, CD22 exhibited the lowest incidence of expression in only 50% of all patients, while CD45, CD58, and CD97 were expressed in 80.9, 59.5 and 92.8%, respectively. Analysis of MRD was performed for each Mo Abs combination at day 0 and day 14 post-induction of chemotherapy by 4-color (FCM). The incidence of MRD was 61.9, 70.6, 60.0 and 55.1% for CD22, CD45, CD58 and CD97, respectively. In B-ALL patients, (CD10/CD19/CD34/CD45)+(CD10/CD19/CD34/CD97) represented the highest incidence of expression of leukemic cells markers with a significant correlation with blasts count, suggesting that these are more specific for MRD detection. Also FCM is relatively cost effective for detection of MRD in ALL patients and its applicability in routine leukemia lab is valuable. MRD evaluation at the end of the induction therapy (i.e. day 35 or 42 according to the different schedules) is advised. Also, Ig/T cell receptor gene rearrangements and gene fusions analyzed by polymerase chain reaction (PCR) are preferred.

Targeting Chronic Myeloid Leukemia Stem Cells through Pharmacological Inhibition of HIF-1α

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell (HSC) disorder due to the 9;22(q34.1;q11.2) reciprocal chromosomal translocation, which results in the generation of BCR/abl "fusion" oncogene and the expression of the BCR/Abl oncoprotein, a constitutively-active tyrosine kinase (TK). To date, chronic-phase CML patients are treated with TK inhibitors (TKi) such as imatinib- mesylate (IM) and dasatinib. However, to eradicate CML, it is necessary to eliminate Leukemia Stem Cells (LSC), which are largely insensitive to TKi. Thus, TKi, rather than curing CML definitively, induce a state of minimal residual disease (MRD) apparently sustained by LSC persistence. Therefore, the identification of non-TKi drugs capable to target and eradicate LSC responsible for MRD and therefore the risk of relapse of disease is of primary importance.LSC, as well as normal HSC, in vivo reside in low oxygen tension areas of the bone marrow (BM) called "HSC niche". Several previous studies from our group and others demonstrate that low oxygen tension maintains the survival and stem cell potential of HSC and LSC, favoring their self-renewal. Interestingly, we also found that the expression of BCR/Abl oncoprotein is suppressed in low oxygen, which well explains the refractoriness of LSC to TKi, provided they manage to survive in the absence of BCR/Abl signaling.Hypoxia-Inducible Factor-1α (HIF-1α) is a key regulator of cell adaptation to low oxygen and of LSC maintenance. This study addresses the in vitro and in vivo effects of the pharmacological inhibition of HIF-1α on the maintenance of CML stem cell potential.After screening several HIF-1α inhibitors, we found that Acriflavine (ACF) induced apoptosis and DNA damage in CML cells and identified ACF as a very effective inhibitor of CML cell survival and growth in low oxygen, as regards the number of viable cells in culture and colony formation efficiency of CML cell lines or human or murine primary cells. Interestingly, ACF did not affect the colony formation efficiency of normal human hematopoietic cells. We also demonstrated in this study that IM and ACF exhibited an at least additive effect as far as the reduction of the total number of viable cells in low oxygen is concerned. Moreover, we demonstrated that low-oxygen incubation of CML cells increased the expression of genes related to stem cell potential, such as Nanog, Sox2 and Oct4, and that ACF treatment reduced their expression. These results on one hand confirmed that hypoxia selects cells with SC potential, on the other hand they demonstrated that ACF treatment reduces their maintenance.Moreover, we evaluated the maintenance of CML stem cell potential using a stem cell assay, demonstrating that ACF suppressed the stem cell potential of CML cell lines or primary cells. On the contrary, IM or Dasatinib, were unable to affect the stem cell potential of CML cells also when administered to primary samples neither TKi-treated before nor carrying primary mutations of BCR/Abl kinase domain. This is in agreement with what observed before by us and others and confirms the refractoriness of LSC to TKi.The effects of ACF in vivo were evaluated using a murine CML model where the disease was induced by a BCR/abl-carrying retrovirus. A 10 day-long ACF treatment was sufficient to attenuate CML development in mice, as evidenced by lower spleen weight, fewer leukemia cells in peripheral blood and BM, and a markedly reduced leukemia cell infiltration into the lungs. Moreover, it was possible to confirm in vivo the effects of ACF on LSC observed in vitro. ACF treatment indeed reduced the maintenance of the Long-Term LSC-containing LSK cell subset (CD34- LSK) in the BM of CML mice. Interestingly and in keeping with the less-severe addiction of HSC to HIF-1α deficiency, we found that non-CML cells were significantly less sensitive to ACF treatment than CML cells. This points to a good therapeutic index of ACF in discriminating leukemic from normal hematopoietic cells.On the basis of all above, the ACF/TKi combination may represent a novel therapeutic approach to target at one time CML cell bulk and LSC, in order to induce remission and prevent MRD of CML. Interestingly, ACF is an already FDA-approved drug for non-oncological uses in humans. DisclosuresNo relevant conflicts of interest to declare.

Towards 'Off-the-Shelf ' Universal Chimeric Antigen Receptor (CAR) T Cells: Mouse Anti-3rd Party Central Memory CD8 Veto Cells Prolong Functional Engraftment of Allogeneic Genetically Modified T Cells

We recently demonstrated that mouse anti-3rd party central memory CD8 veto cells (Tcm) enable engraftment of fully mis-matched T cell depleted bone marrow (TDBM) in sublethally irradiated (5.5 Gy TBI) recipients (Eran et al. Blood 2013). Here we found that Tcm generated from (host x donor)F1 or fully allogeneic donors were able to engraft and sustain their presence in such sub-lethally irradiated mice in the absence of TDBM transplants. Limiting dilution analysis revealed that the reactivity of host T cells (HTC) against donor spleen cells was significantly diminished in the chimeric mice treated with Tcm, whereas reactivity against 3rd party was comparable betweenTcm treated and non-treated mice (Fig. 1). This specific immune tolerance indicated that vetoTcm could potentially enable the use of 'off-the-shelf' allogeneic CAR T cells or any other genetically modified T cells from the same donor.To further investigate this possibility, we tested the ability of Tcmveto cells to enable engraftment of CD8 T cells from OT1 mice, expressing a transgenic T cell receptor (TCR) directed againstovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. To that end, 1x106 OT1 CD8 T cells (H2b) were infused into Balb/c (H2Dd) recipients sublethally irradiated with 5.25 Gy TBI in the presence or absence of different doses of veto Tcm. As shown in Fig. 2, when tested at 2 months post-transplant, OT1 cells could not be detected in the peripheral blood of untreated recipient mice, while they were readily detectable in mice receiving 2x106 Tcm (0.78±0.36), reaching a higher level (1.18± 0.61) upon infusion of 5x106Tcm.To evaluate the functionality of the engrafted OT1 T cells in the chimeric mice, we developed and calibrated a new murine model, using a melanoma B16-cell line of C57BL background that expresses the ovalbumin peptide and the tdTomatomarker (B16-OVA-tdTomato) (Fig 3A). In this model, we tried to mimic a state of minimal residual disease by injecting a small number of B16-OVAtdTomato melanoma cells (0.25x106) into syngeneic C57BL mice; we then treated the mice with sublethal irradiation (6Gy TBI) to simulate treatment of the tumor, but also to clear out space for the T-cell adoptive transfer. Finally, we injected 1x106 naïve OT-1 cells generated on a (Balb x-OT1-CD45.1+RAG2-) F1 background in the presence or absence of Balb/c veto Tcm (5x106), and followed the development of the tumor. We found thatBalb/c vetoTcm prolonged the engraftment of the transgenic donor-derived F1 OT1 CD8 T cells inBalb/c recipients, thereby enabling significantly improved control of tumor growth when tested at day 20 post-transplant ( p<0.05). In contrast non OVA-expressing tumor cells grew at the same rate in all treatment groups, thereby demonstrating the specificity of the anti-tumor effect (Fig.3B).Previous studies demonstrated that Tcmare depleted of GVH reactivity by virtue of their culture with 3rd party stimulators under cytokine starvation. Our present results demonstrate that suchTcm veto cells can pave the way for functional engraftment of CD8 T cells with genetically modified specificity. Taken together, these results provide a proof of concept for the potential clinical use of such veto cells to enable therapy with 'off-the-shelf ' allogeneic CAR T cells. The relative ease of growing humanTcm in large numbers over a short period of time (9-12 days) suggests that it could be possible to harvest byleukapheresis from a single donor, a sufficient number of cells, to support CAR therapy for more than 50 allogeneic recipients.* N.O.G and R.G equally contributed [Display omitted] [Display omitted] [Display omitted] DisclosuresOr Geva:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Characterisation of Molecular Relapse Profiles after Tyrosine Kinase Inhibitor Cessation in Patients Eligible for Treatment-Free Remission Strategies in Chronic Phase CML

Background & aimsTyrosine kinase inhibitors (TKI) still in a majority of cases do not cure chronic phase CML (CP-CML) and leave patients in a minimal residual disease state, requiring the indefinite continuation of TKI. However, in some patients, these agents are able to induce prolonged disease undetectability leading to TKI-cessation strategies and successful treatment-free remissions (TFR) in some. Stable (≥2 years) undetectability can be observed after Imatinib (IM), Nilotinib (NIL) and Dasatinib (DAS) and TKI-cessation strategies have been developed in these 3 settings. In most studies, the overall molecular relapse rate [defined as a loss of MMR, >0.1% BCR-ABL/ABLIS] is ~30-50% requiring the re-initiation of a TKI. The aim of this study is to characterize and compare the molecular relapse profiles in 3 cohorts of patients undergoing IM, NIL or DAS-cessation.MethodsThis is a bicentric observational retrospective analysis on a selected cohort of CP-CML patients fulfilling the following criteria: CP-CML since diagnosis; ≥2 years stable undetectable BCR-ABL (with ≥32,000 ABL copies/sample) evaluated on 4-6 consecutive follow-up samples, on IM, NIL, or DAS; first cessation whatever the reason for TKI prescription was; molecular relapse after TKI cessation defined as MMR loss on 1 occasion. Molecular analyses were performed in 2 reference laboratories belonging to the EUTOS/ELN quality control system. Relevant clinical data were extracted from the 2 databases of these centres, following the rules of our country regulations.ResultsWe identified 29 patients relapsing after IM, 15 after NIL and 14 after DAS cessation with a median age at TKI cessation of 57, 63, and 62.5 respectively (p=0.80). Interestingly, the male/female ratios were 1.41, 0.87 and 0.55 (p=0.38). Sokal score were respectively (L/I/H/NA): 10/12/6/2 for IM, 3/6/6/0 for NIL, 5/4/4/1 for DAS groups (overall p=0.78). Pre-TKI IFN-awas administered in 6 patients in the IM, 4 in the NIL and 1 in the DAS groups (p=0.42). ACA were present at diagnosis in 2, 1 and 1 patients respectively. Two patients had NIL and 7 DAS, in first-line. MR4.5 duration was 37 months for IM, 24.5 months for NIL and 33 months for DAS. Overall TKI duration until TKI cessation were 86 months for IM, 85 months for NIL and 70 months for DAS (p=0.28). The median interval between MR4.5 and TKI cessation was shorter for NIL (25 Months) than for DAS (33 months, p=0.041). The kinetics of relapse are shown in Figure 1 where the evolution of the BCR-ABL ratio by TKI has been smoothed with a polynomial regression.Relapses occurred after a median of 3.94 (0.89-7.2) for IM, 5 (0.69-7.6) for NIL and 4 (2.87-7.52) months for DAS (p=0.86). Kaplan-Meier analysis of the molecular disease-free survival (DFS) according to TKI and since TKI cessation is surprisingly not different (log-rank test p=0.95) between the 3 TKI, suggesting that TKI2 do not slow down the kinetics of relapse. When looking at the KM estimates of molecular DFS since stable MR4.5, it seems longer with DAS than with NIL (long-rank test p=0.018), despite the fact that the duration of MR4.5 before cessation was significantly longer with NIL than with DAS. This might suggest a differential effect of these 2 compounds on the BCR-ABL+ stem cell compartment. TKI were re-initiated after a median of 0.9, 0.8 and 0.75 months after relapse for IM, NIL and DAS. After relapse 6 patients switched from IM to NIL, 1 from NIL to Peg-IFN-a, 4 from NIL to DAS, whereas all DAS restarted the same TKI. MR4.5 was regained after 6.5, 3.9 and 7.41 months in the IM, NIL and DAS groups with a median follow-up of 43, 23.75 and 25 months after relapse respectively. Two patients died, 1 in the IM group from terminal cardiac insufficiency and 1 in the DAS group from metastatic secondary tumor. At last follow-up, patients regained ≥ MMR in all groups except 1 (0.16% at 3 months DAS rechallenge).ConclusionDespite its obvious limitations (relatively low number of patients, retrospective non randomized study) this study characterizes the kinetics of molecular relapse after TKI cessation for TFR and might point out differences suggesting that in these patients the BCR-ABL+ cells responsible for relapse might be different, or that the differential kinase inhibition profile by each TKI induces a different behaviour at relapse. However, these data need to be confirmed ideally in a prospective setting. [Display omitted] DisclosuresNicolini:Ariad, BMS: Consultancy, Speakers Bureau; Novartis: Speakers Bureau. Dulucq:Novartis: Speakers Bureau. Mahon:Pfizer: Honoraria; Ariad: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria. Etienne:BMS: Speakers Bureau; Pfizer: Speakers Bureau; ARIAD: Speakers Bureau; novartis: Consultancy, Speakers Bureau.

749. Escape from Tumor Dormancy Following Gene- or Viro-Therapies Is Mediated by Acquisition of a Phenotype in Which Innate Immune Surveillance Actively Drives Tumor Cell Recurrence

When established (~0.3-0.5cm) primary murine B16 tumors are treated with either cytotoxic gene therapy (HSVtk/GCV), oncolytic virotherapy (intra-tumoral reovirus) or adoptive T cell therapy, a proportion of mice will become apparently tumor free. However, if the animals are observed for prolonged periods of time (>60-100 days), the majority of mice will develop aggressively growing recurrent tumors. Excision of the site of tumor injection during the period of tumor dormancy (no palpable tumor) indicated that residual tumor cells are readily detectable in most mice by histology. Unexpectedly, it was extremely difficult to re-grow the tumor cells from these specks of minimal residual disease in vitro. However, by screening multiple cytokines for their ability to support re-growth of recurrent B16 cells, we identified TNF-α as a major growth factor for recurrent cells. Conversely, treatment of primary B16 populations with TNF-α led to highly significant reductions in tumor cell viability. Similarly, depletion of NK cells, or antibody blockade of TNF-α, from C57Bl/6 mice led to significantly increased tumorigencity of primary B16 tumors. In contrast, depletion of NK cells allowed for decreased tumorigenicity of TNF-α stimulated recurrent B16 cells recovered from mice in a state of minimal residual disease. These data suggest that tumor recurrence may be mediated by a distinct phenotypic switch in vivo. Thus, initially, primary tumor cells are highly sensitive to innate immune cell surveillance through NK cells and TNF-α. However, escape from dormancy in vivo is associated with acquisition of a phenotype in which such TNF-α-mediated immune surveillance actively drives tumor growth. TNF-α growth promoted recurrent cells also expressed high levels of PD-L1 compared to primary tumor cells, suggesting that this may be a further mechanism by which recurrences could emerge in vivo, even in the presence of anti tumor T cell responses. Consistent with this hypothesis, recurrence was significantly inhibited in vivo following gene, viro- or adoptive T cell therapies, by treatment with TNF-α blockade or systemic checkpoint inhibitor therapy.

The Culture Repopulation Ability (CRA) Assay and Incubation in Low Oxygen to Test Antileukemic Drugs on Imatinib-Resistant CML Stem-Like Cells.

Chronic myeloid leukemia (CML) is a stem cell-driven disorder caused by the BCR/Abl oncoprotein, a constitutively active tyrosine kinase (TK). Chronic-phase CML patients are treated with impressive efficacy with TK inhibitors (TKi) such as imatinib mesylate (IM). However, rather than definitively curing CML, TKi induces a state of minimal residual disease, due to the persistence of leukemia stem cells (LSC) which are insensitive to this class of drugs. LSC persistence may be due to different reasons, including the suppression of BCR/Abl oncoprotein. It has been shown that this suppression follows incubation in low oxygen under appropriate culture conditions and incubation times.Here we describe the culture repopulation ability (CRA) assay, a non-clonogenic assay capable - together with incubation in low oxygen - to reveal in vitro stem cells endowed with marrow repopulation ability (MRA) in vivo. The CRA assay can be used, before moving to animal tests, as a simple and reliable method for the prescreening of drugs potentially active on CML and other leukemias with respect to their activity on the more immature leukemia cell subsets.

Prospective Study of Peri-Transplant Use of Sorafenib As Remission Maintenance for FLT3-ITD Patients Undergoing Allogeneic Transplantation

▪Background: FLT3 ITD mutated acute myeloid leukemia remains a therapeutic challenge with very few patients experiencing prolonged disease free survival after standard therapies. FLT3 inhibitors have thus far demonstrated limited single agent activity in this disease. However, administration of FLT3 inhibitors during a state of minimal residual disease and a reconstituting immune system offers a promise of improved disease control.Methods: We designed a prospective trial for patients FLT3 ITD AML undergoing allogeneic transplant, to evaluate the safety, tolerability and outcome of the FLT3 ITD inhibitor sorafenib administered pre- and post-allogeneic transplant. To be eligible, patients needed to be in remission prior to transplant. At the discretion of the treating physician, sorafenib could be started prior to transplant after confirmation of remission and continued until three days prior to the start of the preparative regimen. Otherwise, all patients were started on sorafenib 200 mg twice a day, no earlier than Day +30 and no later than Day +120 post-transplant, after documented engraftment (Plts >50K, ANC >500). Dosing of sorafenib was based on individual patient tolerance: in tolerant patients sorafenib was escalated to 400 mg twice a day while dose reductions occurred for Grade 3 or 4 toxicities in a stepwise manner (200 mg BID, 200 mg QD, 200 mg QoD) until a tolerable dose was identified.Results: We have accrued to date 28 patients with a median age of 54, with FLT ITD AML undergoing allogeneic transplant who were started on sorafenib in the peri-transplant period (8 pre-transplant). Ten patients underwent myeloablative conditioning and received a transplant from a matched related donor (5), a matched unrelated donor (2) or a haploidentical donor (3). Eighteen patients underwent non-myeloablative conditioning and transplant using either a haploidentical donor (10), a matched sibling donor (4), a matched unrelated donor (3) or an umbilical cord blood donor (1). The median duration of post transplant follow up for active patients is 450 days (range, 107-1192 days). The median duration of sorafenib therapy for all patients is 252 days (range, 52-1081). The tolerability of sorafenib in the peri-transplant period varied among patients: 6 patients tolerated the maximum dose of sorafenib at 400 mg twice a day, 14 patients were maintained on 200 mg twice a day, seven patients were maintained on 200 mg daily and one patient was maintained on 200 mg every other day. Six patients died in the post-transplant period with three deaths due to relapsed leukemia and three from transplant-associated toxicity. No primary or secondary graft failures were observed but 9 patients developed grade 2 or higher GVHD (1-4 weeks after starting sorafenib) requiring escalation of immunosuppression therapy. Overall 5 pts have relapsed so far. Three of these patients were off therapy at the time of relapse. One patient relapsed with FLT3 ITD leukemia 117 days after stopping sorafenib at the 24 month planned stoppage time and is now back in remission after resuming sorafenib.Correlative studies evaluating FLT3 inhibition via a plasma inhibitory activity (PIA) assay showed consistent inhibition of FLT3 at all tolerability-determined dosing levels, suggesting that dosing can be safely adjusted in patients having adverse events without loss of FLT3 inhibition. Consistent with this finding was the fact that the adjusted sorafenib trough concentration exposure accounting for the active metabolite (sorafenib + (14.59*sorafenib N-oxide)) was highly variable (70% CV) and not statistically different across all dosing levels. Exploratory analysis of minimal residual disease monitoring, and of T regulatory cell populations and their relation to GVHD in the pre- and post-treatment peripheral blood, are ongoing.Conclusion: Sorafenib is well tolerated in the post-transplant setting irrespective of the conditioning intensity or the donor source. Our findings indicate that sorafenib dosing should be individualized in the post-transplantation setting according to patient tolerability. This approach results in effective in vivo FLT3 inhibition, and yields encouraging survival results, with 15 of 28 patients currently on therapy without relapse. This data supports the further study of sorafenib in a larger cohort to determine its overall effectiveness in preventing FLT3 ITD AML relapse post allogeneic transplant. [Display omitted] [Display omitted] DisclosuresOff Label Use: Sorafenib for off label usage in Acute Myeloid Leukemia.

In the Era of Bortezomib-Based Chemotherapy the Presence of Minimal Residual Disease Predicts Progression Free Survival after Autologous Hematopoietic Cell Transplant

Background: Retrospective studies have shown that achieving minimal residual disease (MRD) by multi-parameter flow cytometry (MFC) is predictive of outcomes in multiple myeloma (MM). Modern induction therapies including bortezomib (V), lenalidomide (R) followed by autologous hematopoietic cell transplant (AHCT) have resulted in substantial improvements in progression free survival (PFS) and overall survival (OS). The impact of bortezomib-based therapy on end of induction chemotherapy MRD status and post-AHCT MRD status is not well defined. We designed a prospective clinical trial (NCT01215344) to study the incidence of MRD negativity at end of induction and following AHCT, and its association with PFS and OS.Patients and Methods: Twenty patients with newly diagnosed, symptomatic MM were enrolled. They received four cycles of VRd induction therapy followed by AHCT. Dose modifications of VRd were controlled during the clinical trial. MRD status by MFC was evaluated at the end of induction chemotherapy and at day 100 post-AHCT. The MFC cut-off to determine MRD negativity was defined as 10-4. Outcomes analyzed included MM disease status, PFS and OS.Results: Three cohorts of patients were identified; patients achieving MRD negative status at the end of induction (cohort 1, 50%), those achieving MRD negative status only after AHCT (cohort 2, 15%) and patients never achieving MRD negative status (cohort 3, 35%). All patients achieving MRD negative status at the end of induction remained MRD negative post-AHCT. There were no significant differences in median age, renal function, disease stage, cytogenetic risk and maintenance chemotherapy post-AHCT between cohorts. The table summarizes characteristics of these cohorts.Patients who never achieved MRD negative status after AHCT had significantly shorter PFS (cohort 3) compared with patients who achieved MRD negative status (cohorts 1 and 2, p=0.008) (figure). The median PFS for cohort 3 was 2.64 years and not yet reached for the other cohorts. There were no significant differences in OS. Median follow-up for survivors was 2.53 years (range, 0.73-4.04).Conclusions: In this study, bortezomib-based therapy resulted in half of patients achieving MRD negative status with induction chemotherapy alone. AHCT improved the depth of response with 30% of patients who were MRD positive after induction therapy converting to MRD negativity following AHCT. Achieving a negative MRD state, pre- and at D100 post-AHCT resulted in improved PFS. These findings were independent of molecular cytogenetics or ISS stage. MRD positive status at day 100 post-AHCT is highly predictive of earlier disease progression, and may help identify patients for alternative management approaches. Delay of AHCT may be considered as a potential management option for patients with MRD negative status at the end of induction therapy, as being studied in the IFM DFCI study (NCT01208662). As some patients with MRD positive disease at the end of induction therapy become MRD negative with AHCT, these patients may benefit from AHCT and this population warrants further investigation.TableDescriptive analysisCohort 1 MRD -/- (%) (n=10)Cohort 2 MRD +/- (%) (n=3)Cohort 3 MRD +/+ (%) (n=7)Test StatisticMedian Age5554600.31ISS Stage III4033140.52DS Stage III8967600.14Median Serum creatinine0.900.600.880.10Serum M-spike2.01.92.10.80Bone marrow plasma cells1444140.18Cytogenetics0.37Standard risk40057Intermediate risk506743High risk10330 [Display omitted] DisclosuresCornell:Prothena: Research Funding. Jagasia:Takeda Inc: Research Funding.

Beyond consolidation: auto-SCT and immunotherapy for plasma cell myeloma.

Autologous hematopoietic cell transplantation (auto-HCT) is the standard consolidation therapy for plasma cell myeloma patients following induction therapy. Auto-HCT improves disease-free survival (DFS), but is generally not curative. The allogeneic HCT experience demonstrated that T-cell immunotherapy can confer long-term DFS. Preclinical and clinical data indicate that myeloma-associated Ags elicit humoral and cellular immune responses (IRs) in myeloma patients. These findings strongly suggest that the immunotherapeutic strategies, including immune checkpoint inhibitors, therapeutic cancer vaccines and adoptive cellular therapies, are promising avenues of clinical research that may be most applicable in the minimal residual disease state following auto-HCT. These strategies are designed to prime or augment antimyeloma IRs and promote a 'host-vs-myeloma' effect that may result in durable DFS. Innovative clinical trials investigating immune checkpoint inhibitors and cancer vaccines have demonstrated that robust immunity against myeloma-associated Ags can be elicited in the setting of auto-HCT. A diverse array of immunotherapeutic strategies have entered clinical trials in myeloma, including PD-1/PD-L1 inhibitors, DC/myeloma cell fusion vaccines and adoptive chimeric Ag receptor T-cell therapy, and further investigation of combinations of immunologic and pharmaceutical agents are expected in the near future. In this review, we will discuss the preclinical data supporting immunotherapy in auto-HCT for myeloma, clinical investigation of these strategies and the future prospects of immunotherapy in pursuit of the goal of curative therapy.

TP53 Alterations Are Frequent in Patients over 60 Years with B-Precursor Acute Lymphoblastic Leukemia (ALL) and Low Hypodiploid/Near Triploid (HoTr) Karyotype; They Correlate with RB1 Deletion and Leukemic Telomere Gain

IntroductionTP53 is the most frequently altered gene in cancer. Until recently data on its frequency and prognostic impact in ALL has been scant, particularly in the elderly. TP53 alteration in the context of telomere shortening (the hallmark of aging) results in telomere attrition and genomic instability with resultant overexpression of telomerase to overcome the genomic crisis.ObjectiveTo determine TP53 alteration frequency and its association with cytogenetic subgroups, key B cell differentiation/cell cycle genes abnormalities and induction therapy response in ALL patients aged ≥60 and its impact on leukemic telomere stateMethodsPatients enrolled in the UK NCRI UKALL14 and UKALL60+ trials aged ≥60 years were assessed at diagnosis for 17p deletions by cytogenetics, TP53 alterations by FISH and/or direct sequencing of hot spots exons 5-8. Data were correlated with IKZF1, CDKN2A/B, PAR1, BTG, RB1, ETV6, EBF and PAX5 gene copy number status, assessed by MLPA (P335-B1). MYC-rearrangement was an exclusion criterion. Minimal residual disease (MRD) was assessed by quantitative PCR (qPCR) for Ig/TCR rearrangements or BCR-ABL1, where applicable. Leukemic DNA telomere length relative to that of remission DNA was assessed by monochrome multiplex (MM) qPCR and expressed as a log2 ratio. Using expectation maximization mixture model a log2 ratio >3.18 defined gain (reflecting telomerase overexpression) and values below this were defined as loss.ResultsThe cohort included 63 patients; characteristics are presented in table 1. Median age was 63 years (range 60–83). TP53 state by cytogenetics and/or sequencing was available in all. Eleven had TP53 alteration (17%); TP53 mutation only, n=3; TP53 deletion only, n=6; TP53 deletion and mutation, n=2. Cytogenetics was available in 60 patients. Nine patients had HoTr (14%). Seven of 9 patients with HoTr had TP53 alteration (78%) versus 4/51 without HoTr (4%); p<0.001. MLPA was done in 57 patients. Those with TP53 alteration had more copy number alterations (CNA)(median 4 versus 1; p<0.01). Twelve (21%) had RB1 deletion; 3 were due to cytogenetically visible 13q abnormalities rather than ‘true’ intragenic aberration. Four of 9 patients with TP53 alteration had RB1 deletion versus 8/51 without TP53 alteration, p=0.07. All other microaberrations were not significantly associated with TP53 alteration. Sixty-two achieved complete remission after 2 cycles of induction. MRD state (n=32) was not impacted by TP53 (2/5 with TP53 alteration and 13/27 without were MRD positive after 2 cycles of induction; p=0.563). Fourteen of 59 (24%) patients relapsed; median of four months from diagnosis (range 2–21), three had TP53 alteration versus 11 without TP53 alteration; p=0.65. Relapse occurred significantly earlier in patients with TP53 alteration (median two months versus eight months; p=0.03). Leukemic telomere state was assessed in 28 patients. Three of 4 patients with telomere gain had TP53 alteration versus 3/24 without; p=0.02ConclusionsThis is one of the largest reports of TP53 frequency in older patients with non-MYC-rearranged ALL. TP53 was deleted and/or mutated in 17%, with highest frequency in those with HoTr (7/9). RB1 deletion was the commonest associated microaberration, noted in over a third of patients. TP53 alteration was associated with earlier relapse but had no impact on induction response. TP53 alteration was notably associated with frequent CNA and telomere gain suggesting a putative potential role of TP53 in genomic instability though sample size for the latter limits assertion of this hypothesis. Mechanistic work to shed insight into this model is currently underway.Table 1: Patient characteristicsTP53 deleted and/or mutated n=11TP53 wild type n=52Age at Diagnosis, median(range)63(60-75)63(60-83)Gender; M:F06:0523:29Phenotype, n (%)· Precursor B cell11 (100)51 (98)· Precursor T cell0 (0)1 (2)Cytogenetics (risk stratification as per Moorman et al. Blood 2007; 109:3189–97), n (%)Standard Risk0 (0)23 (44)t(9;22)1 (9)21 (40)t(4;11)0 (0)2(4)Ho/Tri7 (64)2(4)Complex2 (18)2 (4)Unknown1 (9)2 (4)Morphological Remission after Cycle 2 Induction, n (%)11 (100)51(98)Induction Deaths, n (%)2 (18)15 (29)CNA by MLPA, median (range)4 (2-7)1 (0-2)RB1 deletion, n (%)· Present4 (36)8 (15)· Absent5 (45)43 (83)· Unknown2 (18)1 (2)Leukemic Telomere State· Gain3 (27)3 (6)· Loss1 (9)21 (40)· Unknown7 (64)28 (54) DisclosuresNo relevant conflicts of interest to declare.

Pilot Study of Regulatory T Cell Depletion In The Setting Of Autologous Stem Cell Transplantation For Multiple Myeloma

Although autologous stem cell transplantation (ASCT) is effective for patients with multiple myeloma (MM), disease relapse remains problematic. Despite significant advances in therapeutics for MM, it remains mostly incurable. Following ASCT, a majority of MM patients are in a minimal residual disease state, at which time delivery of immunotherapy may be most effective. Regulatory T cells (Tregs) are a naturally-suppressive CD4+ T cell population (CD4+CD25+FoxP3+ T cells) that expand in MM patients, and strongly inhibit anti-tumor immune responses. In pre-clinical models Treg depletion enhanced the function of tumor antigen-specific T cells. Effective strategies to deplete Tregs in humans are being investigated. We initiated pilot study to test 2 methods of Treg depletion in MM patients undergoing ASCT. In the first approach, an anti-CD25 monoclonal antibody (basiliximab) was administered day +1 following ASCT (in vivo Treg depletion). The second method involved depleting CD4+CD25+ Tregs from autologous stem cell (ASC) grafts with clinical-grade anti-CD25 microbeads and the CliniMACS device (Miltenyi) (ex vivo Treg depletion). A control arm consisted of patients in which no Treg-depleting maneuver was performed. To date, 10 patients with symptomatic MM have been enrolled and randomly assigned to 1 of 3 study arms (arm 1 – standard ASCT; arm 2 – in vivo Treg depletion; arm 3 – ex vivo Treg depletion). Primary endpoints included: a) efficacy of ex vivo Treg depletion from ASC grafts, b) kinetics of Treg depletion and recovery in each study arm, and c) assessment of toxicity associated with Treg depletion. Secondary endpoints included time to engraftment following ASCT and disease response. The overall goal of the study was to identify a superior strategy with regard to depth and durability of Treg depletion. Ten patients (median age 59; range 46-68) have been enrolled. 4 were enrolled onto arm 1, 3 onto arm 2 and 3 onto arm 3. One patient enrolled on arm 2 was removed from study due to ASC mobilization failure. ASC were collected following neupogen and plexifor, and conditioning for ASCT consisted of melphalan (200mg/m2). Typical serious adverse events associated with ASCT were observed in all study arms, with neutropenic fever being the most common (n=2 arm 1, n=1 arm 2 and n=2 arm 3). Autoimmune complications, such as autologous graft-versus-host disease, were not observed. All patients engrafted post-ASCT with normal kinetics. Too few patients have been treated to make conclusions regarding clinical efficacy. Depletion of CD4+CD25+ Tregs from ASC grafts in arm 3 (ex vivo Treg depletion) was effective. As shown in [Figure 1][1] , 90.1% +/- 6% of CD4+25+ Tregs were depleted from 3 ASC grafts following the procedure. Baseline frequencies of peripheral blood (PB) CD4+FoxP3+ Tregs were similar between patients in all arms (p = n.s.). At least a 2-fold reduction in PB Treg frequency was observed at days +28 and + 60 following ASCT ( [Figure 2][2] ). At day +28, mean numbers (+/- SD) of CD4+FoxP3+ PB Tregs were 8.58% +/- 4.52%, 2.48% +/- 1.77%, and 3.28% +/- 1.12% in arms 1, 2 and 3, respectively (p = 0.08 arm 1 vs arm 2; p = 0.05 arm 1 vs arm 3; p = .57 arm 2 vs arm 3). These data suggest that both the in vivo and ex vivo Treg depletion may be effective at mediating effective and durable Treg depletion. ![Figure 1][3] Figure 1 Treg are effectively depleted ex vivo with CD25 microbeads and the Clinimacs device. A FACS plots showing frequency of CD4 +CD25 + Tregs before (left) and after (right) ex vivo Treg depletion procedure (arm 3). B Mean CD4 + CD25 + Treg frequencies before and after ex vivo Treg depletion (*p= 0.05). ![Figure 2][3] Figure 2 Kinetics of Treg depletion following ASCT. Mean (+/- SD) frequency of CD4+FoxP3+ Tregs before and after ASCT in each study arm are displayed. In conclusion, both the in vivo or ex vivo method of Treg depletion appear to be effective based on small patient numbers. Treg depletion in MM patients undergoing ASCT is safe and does not delay engraftment. Additional patients will need to be treated to make conclusions regarding clinical efficacy. Future studies of Treg depletion in combination with other immunotherapeutic strategies are being considered. Disclosures: No relevant conflicts of interest to declare. [1]: #F1 [2]: #F2 [3]: pending:yes

Heat shock protein vaccination and directed IL-2 therapy amplify tumor immunity rapidly following bone marrow transplantation in mice

AbstractTumor relapse is the primary cause of mortality in patients with hematologic cancers following autologous hematopoietic stem cell transplantation (HSCT). Vaccination early after HSCT can exploit both the state of lymphopenia and minimal residual disease for generating antitumor immunity. Here, multiple vaccinations using lymphoma cells engineered to secrete heat shock protein fusion gp96-Ig within 2 weeks of T cell-replete syngeneic HSCT led to cross-presentation and increased survival of lymphoma-bearing mice. To enhance vaccine efficacy, interleukin (IL)-2 was directed to predominantly memory phenotype CD8+ T lymphocytes and natural killer (NK) cells via administration bound to anti-IL-2 monoclonal antibody clone S4B6 (IL-2S4B6). Combination therapy with gp96-Ig vaccination and coordinated infusions of IL-2S4B6 resulted in marked prolongation of survival, which directly correlated with ∼500% increase in effector CD8+ T-cell numbers. Notably, this dual regimen elicited large increases in both donor CD8+ T and NK cells, but not CD4+ T lymphocytes; the former 2 populations are essential for both vaccine efficacy and protection against opportunistic infections after HSCT. Indeed, IL-2S4B6-treated HSCT recipients infected with Listeria monocytogenes exhibited decreased bacterial levels. These preclinical studies validate a new strategy particularly well suited to the post-HSCT environment, which may augment adaptive and innate immune function in patients with malignant disease receiving autologous HSCT.

Minimal residual disease monitoring: the new standard for treatment evaluation of haematological malignancies?

Minimal residual disease (MRD) refers to the small number of malignant cells that remain after therapy when the patient is in remission and shows no symptoms or overt signs of disease. Current treatment protocols for haematological malignancies allow most patients to obtain some form of MRD state, but cure seldom follows and in most cases fatal relapses occur sooner or later, leaving a bitter impression of having won a battle yet lost the war. MRD detection and quantification are used for evaluation of treatment efficiency, patient risk stratification and long-term outcome prediction. Whereas multicolour flow cytometry (MCFC) and polymerase chain reaction (PCR) based methods constitute the two most commonly used techniques for MRD detection, next generation sequencing will certainly be widely employed in the future. As MRD reflects the nature of the malignant disease itself, including its sensitivity to the drug regimens applied, it constitutes the ideal method for surveillance and patient follow-up. The morphological examination of peripheral blood or bone marrow smears, although still an indispensable part of routine laboratory testing, is clearly insufficient for patient management, and clinicians should not ask themselves whether to look for MRD or not, but how and when.

Prophylactic Donor Lymphocyte Infusion to Prevent Relapse after Allogeneic Stem Cell Transplantation

Donor lymphocyte infusion (DLI) has been used to treat disease relapse after stem cell transplantation, but high incidence of graftversus-host disease (GVHD) after traditional DLI, and low response rates with robust relapse in acute leukemia have limited its wide application. Nonetheless, the promising graft versus leukemia effect elicited by DLI makes it a viable option, especially in the state of minimal residual disease after stem cell transplantation. Prophylactic donor lymphocyte infusion has been explored as a more tolerable, less toxic and efficacious option to prevent relapse. This review will summarize the results of the latest prophylactic DLI studies, and discuss our ongoing prophylactic DLI clinical trial after T cell depleted stem cell transplantation in patients with high risk hematologic malignancies.