Doses Of VP-16 Research Articles

Compassionate Use of Emapalumab in Children with Life-Threatening Disorders: Real-World Data

Introduction: Life threatening hyperinflammation occurs in hemophagocytic lymphohistiocytosis (HLH) and during acute rejection of hematopoietic stem cell (HSC) grafts. Both conditions reflect acute activation of the immune system, in part due to overproduction of proinflammatory cytokines, particularly interferon gamma (IFNγ). Emapalumab, a fully human monoclonal antibody that binds to IFNγ, is approved by the FDA for refractory or recurrent primary HLH (pHLH) or in patients intolerant to conventional therapy. Off-label use of emapalumab has been reported for treatment of secondary HLH (sHLH) and for HSC graft rejection. The European Medicines Agency did not approve emapalumab for the treatment of HLH and many other regulatory authorities, including the Israeli Ministry of Health, have yet to approve emapalumab for clinical use. Pending such regulatory approval, emapalumab is available in Israel exclusively though the Managed Access Request Program (MAP) on a compassionate basis sponsored by Sobi (Swedish Orphan Biovitrum AB,Reg. no. 556038-9321). Experience that accumulates through on- and off-label use of emapalumab obtained via MAP enriches the knowledge base regarding the uses of this drug in the real-world clinical setting. Methods: We summarized the data of patients receiving emapalumab at our tertiary pediatric center. The medication was received after application to the MAP sponsored by Sobi International with import approval of the Israel Ministry of Health on a named-patient basis. Results: Seven patients (Pt) aged 2.5 months-17.5 years were treated with emapalumab between 3/2020-6/2023. Patients 1-5 fulfilled HLH-2004 diagnostic criteria. Indications for the use of emapalumab included treatment of HLH in a newborn with severe hepatotoxicity after treatment with the standard HLH-94 protocol (Pt. 3), treatment of acute graft rejection with recurrence of HLH in a patient with pHLH (Pt.1), secondary HSC graft failure and disease relapse in a child with pHLH (Pt. 4), sHLH with multi-organ failure (MOF) requiring extracorporeal membrane oxygenation (ECMO) (Pts 2&5) and HSC primary graft rejection with suspected 2 nd rejection (Pts.6&7). Table 1 summarizes the clinical characteristics of all cases. The patient outcomes were closely related to their clinical status at treatment initiation. Pt.3 was able to reach remission and successfully bridge to SCT without complications after only 4 doses of VP-16 with continuation therapy using only dexamethasone and emapalumab. Pt.4, who experienced relapsed pHLH with concomitant HSC graft loss, attained complete remission with emapalumab, dexamethasone and IVIG, facilitating a successful second HSC graft. Pt. 1 expired after initial graft failure, severe sepsis and relapse of pHLH. Pts. 2&5, despite showing improvement of their HLH criteria after treatment with emapalumab, succumbed to MOF that was already present when they were referred to our center for ECMO support. Following unsuccessful emapalumab treatment for primary graft rejection, Pts. 6&7 underwent successful 2 nd HSC transplants with early emapalumab treatment for suspected re-rejection. Conclusions: Emapalumab is a safe, effective, and well tolerated treatment for various HLH scenarios, and for similar hyper-inflammatory medical conditions. Administration of emapalumab early in the disease course is likely to be more effective as compared to administration of the drug when MOF has occurred or when the HSC graft has already been rejected. Since these diseases are life-threatening, eliminating delays in procurement of emapalumab is essential to expedite treatment before irreversible organ failure ensues.

Analysis of Related Factors of Autologous Peripheral Hematopoietic Stem Cell Mobilization in Patients with Lymphoma and Myeloma

To explore the related factors affecting of autologous peripheral hematopoietic stem cell mobilization in patients with single center lymphoma and multiple myeloma. The clinical total of 30 patients with lymphoma or multiple myeloma who underwent autologous peripheral hematopoietic stem cell mobilization and transplantation in the Affiliated Hospital of Jiangsu University from March 2012 to December 2021 were retrospectively analyzed, including the patients' age, gender, disease type, chemotherapy course, mobilization scheme, collection times, CD34+ cell count, adverse events, days of neutrophil and platelet implantation after transplantation. The related factors affecting to the mobilization efficiency of peripheral blood stem cells was analyzed. The mobilization scheme had a significant effect on the mobilization success rate of CD34+ cells. The mobilization success rate and optimal mobilization rate of intermediate-dose VP-16+G-CSF were higher than that of high-dose VP-16+G-CSF (P<0.05); the mobilization success rate of patients with previous chemotherapy courses ≤4 was higher than that of patients with chemotherapy courses >4 (100% vs 72.22%, P<0.05); the mobilization success rate of lymphoma patients was lower than that of myeloma patients (66.67% vs 94.44%, P<0.05); the mobilization success rate of lymphoma patients who received intermediate-dose VP-16+G-CSF was higher than that received high-dose VP-16+G-CSF patients (100% vs 42.86%, P<0.05). Patients' gender, age, time from diagnosis to mobilization and disease status had no significant effect on the efficiency of stem cell mobilization. Fifteen patients (50%) had febrile neutropenia during stem cell mobilization. There was no statistical difference in the incidence of febrile neutropenia between the two mobilization schemes (P>0.05); the incidence of severe thrombocytopenia in intermediate-dose VP-16+G-CSF group was higher than that in high-dose VP-16+G-CSF group (P<0.05). There was no statistical difference in the time of granulocyte implantation and platelet implantation after stem cell transplantation in patients with different mobilization schemes (P>0.05). Mobilization regime, the number of previous chemotherapy course and disease type affect the mobilization efficiency of stem cells. Intermediate dose VP-16+G-CSF can improve the mobilization efficiency of stem cell in lymphoma patients, but should pay attention to the risk of bleeding.

Optimal chemo-mobilization for the collection of peripheral blood stem cells in patients with multiple myeloma

BackgroundFor successful autologous stem cell transplantation, the collection of a sufficient number of hematopoietic stem cells after induction therapy is essential for transplant candidates with multiple myeloma (MM).MethodsIn this study, we compared the efficacy and safety of stem cell mobilization using cyclophosphamide (CY; 3.0 g/m2 on day 1) or etoposide (VP-16; 375 mg/m2 on days 1 and 2) in patients with MM. Granulocyte-colony stimulating factor (G-CSF, 10 μg/kg/day, subcutaneously) was administered from the onset of neutropenia to the final day of collection.ResultsSixty-five patients were mobilized with a combination of CY and G-CSF, and 63 were mobilized with a combination of VP-16 and G-CSF. All patients were mobilized within 7 months of beginning frontline treatment. The median number of CD34+ cells collected was significantly higher in the VP-16 mobilization group than in the CY mobilization group (27.6 × 106 CD34+/kg vs. 9.6 × 106 CD34+/kg, P < 0.001). The rate of mobilization failure, defined as < 2.0 × 106 CD34+/kg collected in three apheresis procedures, was lower in the VP-16 group than in the CY group (1.6% vs. 10.8%, P = 0.062). Severe infections during the mobilization period were more frequent in the CY group than in the VP-16 group (18.5% vs. 7.9%, P = 0.117).ConclusionIn conclusion, an intermediate dose of VP-16 with G-CSF appears to be an effective and tolerable chemo-mobilization method compared to CY and G-CSF, particularly in cases where use plerixafor in MM is difficult.

Mechanisms of Susceptibility to 11q23 MLL Gene Locus Rearrangements in CD34+ Cells Exposed to Etoposide.

Abstract 185Exposure to the topoisomerase II inhibitor etoposide (VP16) is a significant risk factor in the development of t-MDS/AML. VP16 induces a variety of chromosomal lesions, with the most prominent ones involving the Mixed Lineage Leukemia (MLL) gene on chromosome 11q23. The MLL gene is essential for the maintenance of adult hematopoietic stem cells (HSCs) and progenitors. It is not clear whether the high frequency of MLL gene rearrangements in VP16-treated patients represents increased susceptibility of this locus to VP16-induced damage or a growth advantage of MLL-rearranged hematopoietic progenitors. In the present study we investigated the following: i) the sequence of acquisition of chromosomal lesions in normal CD34+ cells following in vitro exposure to VP16; ii) the specific propensity for development of lesions in 11q23 MLL locus; iii) and the fate of these lesions over time. CD34+ cells were selected from peripheral blood stem cell samples obtained from normal donors (n=5); they were exposed to increasing doses of VP16 (0-40 μM) for 4 hours; washed and cultured in serum-free medium containing growth factors; and assessed for apoptosis, colony forming cell (CFC) assay and chromosomal damage. Chromosome painting was performed using whole chromosome probes for chromosomes 1, 5, 7, 11 and 21, covering 25.2% of total genomic DNA with >100 metaphases scored per dose per time point. Evaluations were performed after 72 hours of culture, representing the 1st cell division after VP16 exposure, and after 144 hours representing 3-4 cell divisions. A dose-dependant decrease in colony formation (71.52 ± 0.99% of controls with 5 mM VP16, and 33.62 ± 4.4%%, with 20 mM VP16 at 72h, p<0.001), and increase in apoptosis (9.12 ± 1.67% with 5 mM VP16, and 39.3 ± 5.06 with 20 mM VP16 at 72h, p<0.001) was observed. The percentage of aberrant metaphases at 1st division was 2.4% with 5 μM and 14.9% with 20 μM of VP16. Both stable aberrations (translocations) and unstable (dicentrics, fragments) aberrations were observed. The percentage of aberrant metaphases was reduced after 3-4 cell divisions (1.4% with 5 μM and 8.7% with 20 μM VP16), and only stable aberrations were present whereas unstable aberrations were eliminated. A higher frequency of aberrations was observed in chromosome 11 compared to the other chromosomes. A 1.5- to 4-fold increase in stable lesions of chromosome 11 relative to other chromosomes was seen at the 1st division and a 2- to 3-fold increase relative to other chromosomes was seen after 3-4 divisions. To assess whether the 11q23 MLL locus was specifically affected by VP16 treatment, we performed interphase FISH with a break-apart probe for the MLL gene (11q23). A break-apart probe for the MYC gene (8q24) was studied as a control. Cells were cultured for three weeks. In preliminary experiments we observed that a higher percentage of cells showed 11q23 abnormalities (9.3%) compared with 8q24 abnormalities (5.4%) at the 1st division. 11q23 abnormalities persisted at high levels in cells (9.9%) cultured for 3 weeks. In contrast, 8q24 abnormalities were markedly reduced (0.4%) after 3 weeks of culture, suggesting selective maintenance of MLL-rearranged cells in culture. In conclusion we show that in vitro exposure to VP16 results in a dose-dependent induction of both stable and unstable chromosomal aberrations in CD34+ hematopoietic progenitors. We observe persistence of stable chromosomal aberrations with particular predisposition to aberrations in chromosome 11. Our results suggest a selective predisposition to 11q23 MLL locus rearrangements that appears to be related to enhanced susceptibility to damage to this locus following VP16 exposure as well as selective maintenance of MLL rearranged cells over subsequent cell divisions. These findings are now being applied to understand the relationship between individual susceptibility to acquisition of VP16-induced 11q23 abnormalities in CD34+ cells and risk of development of t-MDS/AML. Disclosures:No relevant conflicts of interest to declare.

Secondary malignant neoplasms after intensive treatment of relapsed acute lymphoblastic leukaemia in childhood

Purpose To investigate the cumulative incidence of and the risk factors for developing second malignant neoplasms (SMN) in children and adolescents following treatment for relapse of acute lymphocytic leukaemia (ALL). Methods Patients (1376) up to 18 years of age with first relapse of non-B-cell ALL were treated and achieved a 2nd complete remission (CR). The treatment followed trial protocol in five consecutive multicentre trials of the ALL-REZ BFM Study Group between March 1983 and December 2001. The incidence of SMN was analysed, correlated with clinical and therapeutic parameters, and compared to the age-specific incidence rates of cancers as cited in German cancer registries. Results Out of the 1376 patients 21 were diagnosed with SMN including non-lymphoblastic leukaemia/myelodysplastic syndrome ( n = 6), osteo-/Ewing’s-/fibroblastic sarcoma ( n = 4), B-cell ALL/lymphoma ( n = 2), thyroid carcinoma ( n = 2), basal cell carcinoma, adeno carcinoma, squamous cell carcinoma, meningioma, malignant histiocytosis, glioblastoma and anaplastic astrocytoma ( n = 1 each). The overall cumulative risk of SMN at 15 years (median follow-up of 13.1 years) was 1.26% ± 0.38% (SE). SMN was found to be significantly associated with stem cell transplantation (SCT), and high cumulative doses of cranial irradiation, etoposide and cyclophosphamide. In multivariate analysis etoposide (VP16) and cyclophophamide (CY) were found to be independently associated with SMN ( p = 0.047 and 0.002). Compared to the incidence of neoplasm in the age-matched population, there was a 10-fold increase of neoplasia. Conclusions Despite repeated exposure to intense frontline and relapse treatment (including multiagent chemotherapy, cranial irradiation and stem cell transplantation in some patients) the cumulative incidence of SMN was unexpectedly low, though significantly higher than in the general age-matched population. The association of SMN to SCT seemed to be a secondary effect at least partially mediated by exposure to high doses of VP16 and CY given for conditioning therapy.

358: Outcome of hematopoietic stem cell transplantation for pediatric acute lymphoblastic leukemia in third complete remission (CR3): A vital role for graft-versus-host-disease/graft-versus-leukemia effect in survival

Children with acute lymphoblastic leukemia (ALL) who suffer 2 relapses could be salvaged by hematopoietic stem cell transplantation (HSCT) provided they respond to the pre HSCT chemotherapy and enter remission. However, these patients are at very high risk for post HSCT relapse and also at a high risk for transplant related mortality (TRM). Our objective, herein, was to review the outcome of children (0-18years) with ALL who received allogeneic HSCT in third complete remission (CR3) at our institution. From January 1994 – August 2005, twenty-two consecutive children in CR3 received HSCT in our instituation. Conditioning regimens included single dose VP16 (60mg/kg IV over 4 hours) and fractionated total body irradiation (TBI; 1200cGy) in six fractions over 3 days (VP16/TBI) in 10 patients and cyclophosphamide 50mg/kg IV over 1 hour daily for 4 days followed by the same dose of TBI (CY/TBI) in 12 patients. Almost all children received cyclosporine A and a short course of methotrexate for graft-versus-host disease (GVHD) prophylaxis, and all patients were in complete morphological remission prior to HSCT. Median age was 8.4 years (range 3-15.4). Donor sources were as follows: matched sibling donor (MSD), 8; matched unrelated donor (MUD) 6; one antigen mismatch related donor (MMRD) 4; one antigen mismatched unrelated donor (MMUD) 3 and one patient received 1 antigen mismatched cord progenitor stem cells. White cell engraftment was successful at a median of 18 days (range 9-29). Ten patients died of TRM, seven relapsed, one died from other causes and four patients are long term survivors at a median follow up of 3.7 years (range 1-10.2). All patients who did not develop clinical acute or chronic GVHD relapsed and died. Event free survival was (EFS 19% ± 4%). Three out of the 4 survivors received MMUD and all 4 survivors had moderate to severe acute GVHD and three had chronic GVHD, limited in two and extensive in one. Conclusion: Children with ALL in CR3 receiving HSCT are extremely high risk for relapse and transplant related mortality. These children have already relapsed twice and demonstrated chemotherapy resistance and GVL/GVHD plays a key role in leukemia eradication. Although, TRM is high in such patients and GVHD could potentially increase TRM, there are no survivors without GVHD and exploring means of inducing GVHD by reduction of immunosuppressive medications or other means of immunotherapy should be considered in these patients.

Important Role for Absolute Lymphocyte Count in Predicting Risk of Relapse in Pediatric Acute Lymphoblastic Leukemia Post Allogeneic Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is currently an established treatment for children with relapsed or high risk acute lymphoblastic leukemia (ALL). Relapse is a major risk post HSCT and an important cause of treatment failure with abysmal outcome. An important therapeutic mechanism of allogeneic HSCT is the immune-mediated destruction of recipient leukemic cells by donor lymphocytes, the graft-versus-leukemia-effect (GVL). Our hypothesis was to determine if delayed lymphocyte recovery measured by the absolute lymphocyte count (ALC) at two time points post HSCT at day 21 and day 30 correlates with leukemia relapse. We reviewed 136 consecutive paediatric patients with ALL who received allogeneic HSCT between 1994 and 2005 in the Hospital for Sick Children, Toronto, Canada. All patients were in complete morphological remission prior to HSCT and remission status at time of HSCT were as follows; Complete remission 1 (CR1, n=36); Complete remission 2 (CR2, n=79); Complete remission 3 (CR3, n=21). Conditioning regimens included single dose of VP16 (60mg/kg infused over 4 hours) and fractionated total body irradiation (TBI; 1200cGy) in six fractions over 3 days (VP16/TBI) in 49 patients (1994–1998) and cyclophosphamide 50mg/kg infused over 1 hour daily for 4 days followed by the same dose of fractionated TBI (CY/TBI) in 83 patients (1999–2005) and 4 patients received other conditioning regimens. Fifty-six patients received matched sibling donor (MSD), 12 patients received one antigen mismatched related donor (MMRD), 64 received unrelated donors and 4 patients received cord progenitor stem cells. Two groups were identified based on absolute lymphocyte count at day 21 and day 30 post HSCT. Patients with absolute lymphocyte count <0.3×109/L at day 21 (n=104) had more than 5 times risk of relapse compared to those with ALC count >0.3 × 109/L (n=32) (Hazard ratio 0.19; P=0.0004). Patients with ALC <0.3×109/L (n=48) at day 30 were more than twice likely to relapse compared to those with ALC >0.3×109/L (n=88) (Hazard ratio 0.46; P=0.01).Conclusion: ALC, at day 21 and day 30 post HSCT identifies a patient population of pediatric ALL with delayed lymphocyte recovery and a significant risk of relapse post HSCT. Such patients could be considered for early intervention targeted to prevent relapse such as reduction of immunosuppressive therapy or other means of immunotherapy to enhance the graft versus leukemia effect.

Outcome of Hematopoietic Stem Cell Transplantation for Pediatric Acute Lymphoblastic Leukemia in Third Complete Remission (CR3): A Vital Role for Graft-Versus-Host-Disease/Graft-Versus-Leukemia Effect in Survival.

Children with acute lymphoblastic leukemia (ALL) who suffer 2 relapses could be salvaged by hematopoietic stem cell transplantation (HSCT) when a suitable stem cell source is available provided they respond to the pre HSCT chemotherapy and at least enter morphological remission. However, these patients are at very high risk for post HSCT relapse and also at a high risk for transplant related mortality (TRM). Our objective, herein, was to review the outcome of children (0–18years) with ALL who received allogeneic HSCT in third complete remission (CR3) at our institution. Between January 1994 – August 2005, twenty-two consecutive children in CR3 received HSCT in the Hospital for Sick Children, Toronto, Canada. Conditioning regimens included single dose of VP16 (60mg/kg infused over 4 hours) and fractionated total body irradiation (TBI; 1200cGy) in six fractions over 3 days (VP16/TBI) in 10 patients (1994–1998) and cyclophosphamide 50mg/kg infused over 1 hour daily for 4 days followed by the same dose of fractionated TBI (CY/TBI) in 12 patients (1999–2005). Graft-versus-host disease (GVHD) prophylaxis included cyclosporine A and a short course of methotrexate for the majority of patients, and all patients were in complete morphological remission prior to HSCT. Median age was 8.4 years (range 3–15.4). Donor source was as follows: matched sibling donor (MSD), 8; matched unrelated donor (MUD) 6; one antigen mismatch related donor (MMRD) 4; one antigen mismatched unrelated donor (MMUD) 3; and one patient received 1 antigen mismatched cord progenitor stem cells. White cell engraftment was successful in all patients at a median of 18 days (range 9–29). Ten patients died of TRM, seven relapsed, one died from other causes and four patients are long term survivors at a median follow up of 3.7 years (range 1–10.2). All patients who did not develop clinical acute or chronic GVHD relapsed and died. Event free survival was (EFS 19% ± 4%). Three out of the 4 survivors received MMUD and all 4 survivors had moderate to severe acute GVHD and three had chronic GVHD, limited in two and extensive in one.Conclusion: Children with ALL in CR3 receiving HSCT are extremely high risk for relapse and transplant related mortality. These children have already relapsed twice and demonstrated chemotherapy resistance and GVL/GVHD plays a key role in leukemia eradication. Although, TRM is high in such patients and GVHD could potentially increase TRM, there are no survivors without GVHD and exploring means of inducing GVHD by reduction of immunosuppressive medications or other means of immunotherapy should seriously be considered in these patients.

Comparative outcome of hematopoietic stem cell transplantation for pediatric acute lymphoblastic leukemia following cyclophosphamide and total body irradiation or VP16 and total body irradiation conditioning regimens

To compare the outcome of hematopoietic stem cell transplantation (HSCT) in pediatric acute lymphoblastic leukemia (ALL) conditioned with two different regimens: (1) single dose of VP16 (60 mg/kg over 4 h) and total body irradiation (TBI; 1200 cGy, in six fractions) or (2) Cyclophosphamide 50 mg/kg over 1 h daily for 4 days followed by the same dose of TBI. One hundred and seven children with ALL received fully matched HSCT from 1990 to 2003 in the Hospital for Sick Children, Toronto. All received cyclosporin A and a short course of methotrexate for graft-versus-host disease (GVHD) prophylaxis. The VP16 group, there were 36 matched related donor transplants (MRD) and 26 matched unrelated donor transplants (MUD), and in the cyclophosphamide group there were 23 MRD and 22 MUD transplants. Neutrophil engraftment occurred at a median of 18 and 17 days for the VP16/TBI and the CY/TBI groups, respectively. The 3 year event-free survival and overall survival were 47 +/- 7 and 55 +/- 7% for those receiving VP16/TBI, and 51 +/- 8 and 53 +/- 8% for the CY/TBI group. There were no significant differences in the prevalence of acute or chronic GVHD and transplant-related mortality between the two groups. Both VP16/FTBI and CY/FTBI regimen are equally effective regimens.

A phase I and pharmacokinetic study of the selective, non-peptidic inhibitor of matrix metalloproteinase BAY 12-9566 in combination with etoposide and carboplatin

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade the extracellular matrix during the processes of invasion, metastasis and angiogenesis. BAY 12-9566 (BAY) is a selective, non-peptidic biphenyl inhibitor of MMPs, with nanomolar inhibitory activity against MMP-2, -3 and -9, and anti-invasive, anti-metastatic and anti-angiogenic activity in a variety of tumor models. This phase I study of oral BAY was conducted to evaluate the safety and pharmacokinetics of BAY when administered in combination with etoposide (VP-16) or in combination with VP-16 and carboplatin (CBDCA) in subjects with advanced cancer. The first cohort of patients (n=8) received a cycle of VP-16, 60 mg/m, followed 1 week later by a fixed daily oral dose of BAY, 800 mg b.i.d., to which three potential possible doses of VP-16 (low dose: 60 mg/m; mid dose: 90 mg/m; high dose: 120 mg/m) were added every 3 weeks as tolerated. The second cohort (n=5) received VP-16 (120 mg/m) and CBDCA (AUC=5) followed 1 week later by a fixed daily oral dose of BAY (800 mg) b.i.d., to which VP-16 (120 mg/m) and CBDCA (AUC=5) were added. Dose-limiting toxicity (DLT) was defined as toxicity grade 3 or above. Maximum tolerated dose was declared if two or more patients experienced DLT. A performance status of 0-2 and acceptable organ function were required for eligibility. Plasma concentrations of BAY and VP-16 were measured to investigate pharmacokinetic interactions. Eight eligible patients with a variety of tumor types (median age 64 years, range 44-76) were enrolled in the first cohort, six of who whom completed all three levels of VP-16. Progressive disease occurred in five of the eight patients; three patients continued on study with treatment. Drug level and pharmacokinetics analysis of BAY and VP-16 were also determined. The combination of BAY and VP-16 was tolerable in the first cohort, permitting enrollment of the second cohort. In the second cohort (n=5), the combination of BAY, VP-16 and CDBCA was intolerable at the doses used due to excessive hematologic toxicity in the first five patients enrolled. Pharmacokinetics and toxicity analysis was performed for this group of patients. Only Level 1 of treatment was completed for Cohort II. At this point the study was halted due to toxicity and the results of an interim analysis that failed to demonstrate sufficient clinical activity of this compound in other clinical trials. We conclude that the combination of BAY and VP-16 was well tolerated. However, the combination of BAY, VP-16 and CDBCA produces significant hematologic toxicity. Findings from this study may help to direct further studies with other inhibitors of MMPs.

Bone Marrow Replacement with a Clone Harboring Novel t(4;11)(p12;q23) Involving MLL during Neuroblastoma Treatment Is Not Associated with Leukemia.

Epipodophyllotoxins, anthracyclines and other chemotherapeutic DNA topoisomerase II poisons are associated with MLL-rearranged leukemias and, less often, leukemias with different balanced translocations. Every MLL translocation identified so far in patients has been associated with leukemia and, where tested, murine models have established that the der(11) protein products are leukemogenic. We here describe a highly novel t(4;11)(p12;q23) that emerged in the bone marrow that confers a proliferative advantage but not a leukemia phenotype in a patient with stage 4 neuroblastoma (NB) following N8 therapy, which consisted of 3 cycles of cyclophosphamide/doxorubicin/vincristine, 2 cycles of cisplatin (P)/etoposide (VP), surgery, local radiation, anti-GD2 monoclonal antibody (3F8), myeloablative therapy (thiotepa, carboplatin, topotecan) plus autologous PBSC transplant (harvested after 1 cycle of PVP), followed by 7 more cycles of 3F8 plus GM-CSF, 4 cycles of oral VP, and 5 cycles of accutane. Cumulative doses of doxorubicin and VP were 225 mg/m2 and 5.4 g/m2 (4.2 g/m2 orally), respectively. Seventeen months after NB diagnosis (11.5 months after transplant) at completion of oral VP, surveillance FISH showed t(4;11)(p12;q23) in 10.5% of marrow cells. The patient is now 32 months from NB diagnosis and 1 year off all therapy. Although both karyotype and FISH showed t(4;11) in 82–100% cells in 7 serial subsequent marrows, all had normal morphology and there is no clinical evidence of leukemia. Molecular analyses of sequential bone marrow samples were undertaken to characterize the translocation. Southern blot analysis of the MLL bcr in BamHI and BglII digested DNAs showed two rearrangements consistent with both derivative chromosomes. A new BglII-based reverse panhandle PCR identified the der(4) breakpoint junction sequence in a 3.2 kb BglII genomic rearrangement and provided the sequence to isolate the der(11) genomic breakpoint junction by clonotypic PCR. The partner DNA was a novel gene localized to chromosome band 4p12. The translocation fused intron 8 of MLL with intron 8 in the new partner gene, and occurred with a loss of 8–13 bases from MLL and 31–36 bases from the partner gene. Both breakpoint junctions contained short homologous sequences suggesting NHEJ. By clonotypic nested PCR (sensitivity 1 cell in 104–105 cells) the translocation was absent at NB diagnosis, and was first detectable at the completion of oral VP (16 months after neuroblastoma diagnosis). RT-PCR revealed in-frame der(11) and der(4) fusion transcripts. Real-time PCR analysis of known MLL target genes and differentially expressed genes in MLL-rearranged leukemias revealed low levels of HOXA4, A5, A7, A9, FLT3, MEIS1, and PBX3 compared to other treatment-related leukemias. Temporal emergence of this translocation after the high total VP dose is of interest because others recently found that cumulative doses of VP >6 g/m2 carry an especially high risk of leukemia, raising concern about the role of the oral VP in the genesis of the translocation. This study indicates that the different partner genes of MLL do not confer the same leukemia potential. Because the t(4;11)(p12;q23) confers a proliferative advantage but is insufficient for leukemia, we designated the new partner gene MIFL (MLL Insufficient for Leukemia), however it remains to be determined whether any relevant secondary alteration will result in leukemic transformation.

Outcome of Stem Cell Transplantation for Pediatric Acute Lymphoblastic Leukemia. A Comparison of Cyclophosphamide and Total Body Irradiation Versus VP16 and Total Body Irradiation Conditioning Regimens.

Stem cell transplantation (SCT) has become the standard therapy for children with relapsed acute lymphoblastic leukemia (ALL) or certain high-risk features at diagnosis. The authors report their experience with SCT for 108 children with ALL conditioned with 2 different regimens over 14 years period. Children (0–18y) who received fully matched-related (MRD) or fully matched-unrelated (MUD) for a diagnosis of ALL in the Hospital for Sick Children, Toronto between 1990–1998 were conditioned with a single dose of VP16 (60mg/kg infused over 4 hours) and fractionated total body irradiation (TBI; 1200cGy) in six fractions over 3 days (VP/TBI). Between 1998–2003, children with ALL were conditioned with cyclophosphamide 50mg/kg infused over 1 hour daily for 4 days followed by fractionated total body irradiation (TBI; 1200cGy) in six fractions over 3 days (CY/TBI). All children received cyclosporine A and a short course of methotrexate for graft versus host disease (GVHD) prophylaxis.Results: Sixty-two children with ALL were conditioned with VP16/TBI, 36 MRD transplantation and 26 MUD transplantation. Median age at SCT was 9.4y (range 0.8–19.4). Remissions status at SCT were; complete remission (CR) 1 (n=17), CR 2 (n=32), and CR 3 (n=12) and one patient received SCT in relapse. Forty-six patients were conditioned with CY/TBI, 23 MRD transplantation and 23 MUD transplantation. Median age at SCT was 9.2y (range 1–17.5) Remissions status at SCT time were; CR 1 (n=11), CR 2 (n=29), and CR 3 (n=6). Neutrophils engraftment (defined as absolute neutrophil count (ANC) of > 0.5 x 109/l for 2 consecutive days) occurred at 19d (range 10–28d) and 17d (range 10–28d) for the VP16/TBI and the CY/TBI groups, respectively. Two patients failed to engraft in the VP16/TBI group, one required re- conditioning with cyclophosphamide and ATG and a second dose of stem cells from the same donor, the other patient sustained a fatal intracranial hemorrhage. There was no significant difference in three years event free survival (EFS) and overall survival (OS) between the groups with EFS and OS of 47 ± 7%, 55 ± 7% for the VP16/TBI and 51 ± 8%, 53 ± 8% for the CY/TBI group. There was no significant difference in the incidence of acute or chronic graft versus host disease (GVHD), transplant related mortality (TRM) between the 2 groups.Conclusion: Both VP16/TBI and CY/TB regimen are equally effective preparative regimens for SCT in pediatric ALL.

Oral Low Dose Etoposide in the Treatment of Macrophage Activation Syndrome.

Background: Macrophage activation syndrome (MAS) in adults, also called reactive haemophagocytic syndrome or haemophagocytic lymphohistiocytosis, is the result of inappropriate proliferation and activation of the mononuclear phagocytic system secondary to an underlying disease. Persistent high levels of inflammatory cytokines with impaired cytotoxic capacity of T-lymphocytes and natural killer cells seem to be the main trigger of the syndrome. There are a variety of diseases that can lead to fulminant uncontrolled MAS, mainly viral infections and conditions of immunologic dysregulation such as Systemic Lupus Erythematodes, Morbus Still and neoplastic disorders. Despite the variety of associated diseases, the clinical presentation is uniform with high fever, splenomegaly, lymphadenopathy, hepatomegaly, rash and neurologic abnormalities. MAS is associated with substantial morbidity and mortality. Beside the clinical presentation, extreme elevation of plasma markers for macrophage hyperactivation are mandatory in diagnosing and monitoring MAS (hyperferritinemia, and extreme elevation of neopterin and sIL2R), whereas the phenomenon of hemophagocytosis (±cytopenias) is more variable. Different therapeutic strategies including high-dose steroids, intravenous immunoglobulin (IVIG) [Emmenegger U, et al. American Journal of Haematology 2001; 68:4–10] and intravenous etoposide (VP16)[Imashuku S, et al. Journal of Clinical Oncology 2001; 19:2665–267] have been reported to induce remissions, but tend to be incomplete and of short duration. These treatments are mainly based on studies in children with familial haemophagocytic lymphohistiocytosis and have considerable side-effects. The optimal treatment-approach in adults with MAS remains unclear. Faced with high mortality of MAS at our institution, we accepted VP16 as a paticularly important component of therapy but were struck by fast and live threatening relapses during neutropenic episodes following classical pulse-application of VP16 (2x150mg/m²/d x 2/week). We initiated oral low dose VP16 in refractory RHS and observed substantial effects.Methods and Patients: Our treatment concept was that of uninterrupted macrophage mitigation by continuous low-dose oral VP16 after a first un- or only partially successful episode of high-dose methylprednisolone or IVIG. In the present study we describe the effect of low dose oral VP16 after a first episode treated with high-dose methylprednisolone or IVIG in 3 patients with relapsing or resistant RHS. Close monitoring of cytokine activation was performed by serial measurements of ferritin, sIL2R, transcobalamin II and neopterin (at least twice weekly). 2 of our patients suffered from recurrent MAS in the context of chronic active Epstein-Barr virus infection, 1 patient showed 2 episodes of MAS secondary to Morbus Still. VP16-treatment was dosed at diurnal 50mg for 3 days followed by 15–20mg daily avoiding neutropenic episodes.Results: During the time of observation (4 years) we have ascertained a total of 9 episodes of MAS, 3 of them classified as severe (admission to intensive care unit). 4 episodes of MAS have been managed by oral VP16 exclusively. Decrease of cytokine activation and clinical outcome in episodes treated with oral VP16 proofed to be superior to the other treatment-strategies.Conclusion:Compared to steroids and IVIG, strictly monitored long-term low-dose oral VP16 (for several weeks to months) seems to be a safe and most effective treatment strategy in patients with life-threatening reactive MAS.

Efficacy of etoposide, cyclophosphamide, and total body irradiation in allogeneic bone marrow transplantation for adult patients with hematological malignancies.

A combination of fractionated total body irradiation (TBI) with etoposide (VP-16) and cyclophosphamide (CY) as a preconditioning regimen (VP/CY/TBI) has been reported to be safe and effective for both adults and children undergoing allogeneic bone marrow transplantation (allo-BMT). However, the reported doses of VP-16 were different. We evaluated the efficacy and safety of a VP-16 (at less than the usual dose)/CY/TBI regimen for adults with hematological malignancies who are required to receive allo-BMT. Thirty-eight patients received VP-16, CY and TBI (VP/CY/TBI) as a preconditioning regimen for allo-BMT. Twenty-one patients were in first complete remission (1CR), six patients were in second remission (2CR) and 11 patients were in non-remission status (non-CR) before allo-BMT. These patients received allo-BMT from related donors (n=14) and unrelated donors (n=24). The preconditioning regimen consisted of VP-16 (15 mg/kg/d for 2 d), CY (60 mg/kg/d for 2 d) and 12 Gy TBI in six fractions for 3 d. Two patients died on day 30 after transplantation. The median follow-up period for all patients was 35.0 months (range 0.8-159.6 months). At the time of analysis, 10 patients had died. Seven of those 10 patients died because of relapse. The estimated 5-yr disease-free survival (DFS) rates for all cases and acute myelogenous leukemia and acute lymphoblastic leukemia cases were 73.6, 66.7 and 100%, respectively. The estimated 5-yr DFS rates for 1CR, 2CR and non-CR cases were 90.5, 83.3 and 40.9%, respectively (p < 0.05). Based on these findings, we suggest that a VP/CY/TBI regimen is effective and safe for adult patients with hematological malignancies in 1CR and 2CR.

Evaluation for the Development of 11q23 Rearrangements in Lymphoma Patients Treated with a High Dose VP-16 and Cyclophosphamide Salvage Regimen

Patients with relapsed or refractory lymphoma often require treatment with aggressive chemotherapy. At McGill University, a combination of high dose VP-16 and cyclophosphamide (VP-CY) is commonly used as a salvage regimen. In recent years, cytogenetic abnormalities of the long arm of chromosome 11 at band 23 (11q23) have been linked to the use of VP-16, and may be associated with secondary myelodysplastic syndrome or acute leukemia. Therapy related 11q23 anomalies have not been widely studied in lymphoma patients. We have identified and reviewed the course of 107 patients who have been treated with VP-CY. Thirty-five patients remain alive and 21 consented to participate in our study. Patient bone marrows were studied morphologically, cytogenetically and molecularly, to identify any new changes that may have developed over the course of their treatment, with a special emphasis on the search for 11q23 rearrangements. Mean time between VP-CY treatment and marrow evaluation was 3.6 years. Of the 21 patients, 5 had Hodgkin's disease (HD) and 16 had non Hodgkin's lymphoma (NHL). They received a total of 30 cycles of VP-CY. Response rate was 100%, with 16 complete and 5 partial responses. Eighteen patients later underwent autologous stem cell transplantation. At the time of study, 19 of the patients were disease free and 2 were in relapse. On morphological analysis, 12 marrows appeared normal and 6 showed mild dyserythropoiesis. Standard cytogenetics was done to examine for any new chromosomal translocations or deletions. All cytogenetic studies yielded normal results. Molecular analysis by Southern blot was done on 15 patients in a search for 11q23 rearrangements, including the partial tandem duplication of ALL-1. All molecular studies were normal. We conclude that the use of VP-CY, given in our treatment schedule, does not appear to be associated with an increased risk of developing 11q23 rearrangements.

Molecular cytogenetic analysis in mouse sperm of chemically induced aneuploidy: studies with topoisomerase II inhibitors

The ability of two topoisomerase II (topo II) inhibitors, etoposide (VP-16) and merbarone (MER), to induce meiotic delay and aneuploidy in mouse spermatocytes was investigated. The progression from meiotic divisions to epididymal sperm was determined by injecting male mice with 5-bromo-2′-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. At 20–24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). It was found that VP-16 (50 mg/kg) treatment induced a meiotic delay of about 24 h. A significant reduction of BrdU-labelled sperm was observed at 22 days compared to the controls (VP-16 group: 14.20%; controls: 41.10%, P<0.001). At 23 and 24 days, there were no significant differences between the VP-16 and the control groups. MER (80 mg/kg) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated with 12.5, 25 and 50 mg/kg VP-16 or 15, 30 and 60 mg/kg MER. Sperm were sampled from the Caudae epididymes 24 days after VP-16 treatment or 22 days after MER treatment. Significant increases above the concurrent controls in the frequencies of total hyperhaploid sperm were found after treatment with 25, 50 mg/kg VP-16 (0.074 and 0.122% versus 0.052%) and after treatment with 60 mg/kg MER (0.098% versus 0.044%). Furthermore, significant increases in the frequencies of diploid sperm were found after treatment of mice with all three doses of VP-16 (0.024, 0.032 and 0.056% versus 0.004 and 0.00%, respectively) and with 30 and 60 mg/kg MER (0.022 and 0.05% versus 0.004 and 0.002%, respectively). All dose responses could be expressed by linear equations. The results indicate that cancer patients may stand transient risk for siring chromosomally abnormal offspring after chemotherapy with these topo II inhibitors.

Chemotherapy-induced immunosuppression and reconstitution of immune function

In the cancer patient, there are a variety of factors that favor the suppression of the immune system, including the cancer itself (especially, malignancies of B and T lymphocytes), high dose chemotherapeutic agents, and corticosteroids. In addition, by its very nature, the myeloablative conditioning regimen used for stem-cell transplantation is immunosuppressive. It is important that immune function is restored so that the patient can resist infection, but it may be equally important to reconstitute immunity due to the potential added benefit of an anti-leukemic response. It is the intent of this commentary/review to discuss the effects of chemotherapy on selected immune cell populations and approaches to reconstitute immune function following chemotherapy. In this issue, Slater et al. [1] illustrate the double-edged sword of high dose chemotherapy. Their laboratory investigations demonstrate the cytotoxic efficacy, yet immunosuppressive effects, of high dose topoisomerase II inhibitor VP-16. As a result, treating L1210 tumor-bearing mice with 20 mg/kg VP-16 resulted in a relatively high 60 days survival rate, however, cured mice could not resist a L1210 tumor challenge. In contrast, when tumor-bearing mice were treated with 3 or 5 mg/kg VP-16, correspondingly fewer mice survived, but most survivors were able to reject a tumor challenge. Mitogenic assays revealed a suppressed proliferation potential of splenic lymphocytes treated with the higher dose of VP-16. These investigations exemplify the opposing effects of high dose chemotherapy, i.e. tumor cytotoxic efficacy coupled with immunosuppression. In discussing the effects of chemotherapy on immune function, it is important to differentiate between the terms immunosuppression and myelosuppression [2]. Many chemotherapeutic agents are myelosuppressive in that they inhibit bone marrow production of blood cells and platelets

Enhancement of leukemia rejection by mice successfully treated for L1210 leukemia due to low dose compared to high dose VP-16

We have earlier shown that VP-16 combined with Cyclosporin A (CsA) produces tumor specific immunity to L1210 leukemia in BDF/1 mice [Slater LM, Sweet P, Stupecky M, Reynolds JT. Cyclosporin A/VP-16 produced immunity to L1210 leukemia: the participation of cytotoxic CD 8 T-lymphocytes, Clin Immunol Immunopathol 1995;75:239–45]. Our current studies, designed to determine the role of VP-16 independently of CsA in this effect show that increased dose intensity of VP-16, in the absence of CsA, improves the frequency of 60 day survival of treated mice but impairs the ability of 60 day surviving mice to reject L1210 leukemia challenge. This impairment is associated with progressive diminution of mitogen responses by spleen cells harvested from tumor free VP-16 treated mice.

Etoposide as a virocidal anticytomegalovirus therapy: intravitreal toxicology and pharmacology in rabbits.

Although Cytomegalovirus (CMV) retinitis is now a common intraocular infection, current therapy is only virostatic so ongoing treatment is required. Etoposide was found to be virocidal for CMV in laboratory experiments and it might prove to be beneficial clinically. We investigated the toxicity and intraocular concentration of etoposide (VP16) and its new analogue etoposide-phosphate (VP16P) following intravitreal injections in rabbit eyes. First a sequential dose-response was assessed with flash electroretinogram for both eyes of light- and dark-adapted rabbits (n = 7; one rabbit for each dose) over a range of light intensities before and after intravitreal injection of VP16 or VP16P to one eye; the other eye was injected with normal saline as a control. A multidose study was then performed on four rabbits. A non-toxic dose of VP16P (50 or 75 g) was injected into the vitreous of one eye on four occasions 1 week apart. A photopic electroretinogram was performed before the first injection and 6 weeks after the last injection. All the eyes from the electroretinogram studies were fixed in formalin, placed in paraffin, then stained with haematoxylin and eosin and examined under a light microscope. To determine the time-course of the intraocular concentrations of VP16P a sequential pharmacokinetic study was performed using a further 12 rabbits. Each rabbit was injected with 50 g VP16P to one eye and 75 g VP16P to the other eye. Three of these rabbits were killed at 1, 3, 6 and 9 h after injection. Samples of vitreous were assayed for both VP16 and VP16P using HPLC. An in vitro dose response assay was performed using third-passage bovine retinal pigment epithelial (RPE) cells cultured in Dulbecco's modified Eagles medium with fetal calf serum. The effect of a log-dose increment of VP16P on the RPE cell proliferation was assessed using tritiated thymidine incorporation. The electroretinogram studies suggested that VP16 was toxic even with the 10 g dose. For VP16P a toxic effect was noted following injection of a single dose greater than 100 g. Multiple injections of 50 or 75 g VP16P did not produce a toxic response. Histological examination demonstrated significant abnormality only with the 500 g dose of VP16 or VP16P. VP16P was rapidly metabolized to VP16 in the eye, producing concentrations of 2.0 g/mL or more for up to 9 h following a 75-microg dose. This suggests that the electroretinogram findings following VP16 injections were confounded by a toxic effect of the ethanol solvent (which is absent from the VP16P preparation). VP16P was quite potent, the ID50 was about 0.1 g/mL for bovine RPE cells in the in vitro assay. These results indicate that multiple 75-gVP16P intravitreal injections were not toxic to the rabbit eye and provide a therapeutic intraocular concentration for up to 9 h after the injection.

HCF-dependent nuclear import of VP16.

Transactivation by VP16 requires the formation of a multicomponent complex, the TAATGAAAT recognition factor complex (TRF.C), that contains in addition to VP16, two cellular proteins, Oct-1 and HCF. HCF binds directly to VP16 and this promotes subsequent interaction of the VP16-HCF complex with the POU DNA-binding domain of Oct-1 and selective assembly onto target sites. Here we demonstrate a novel role of HCF in the intracellular compartmentalization of VP16. We show that while VP16 does not contain a consensus nuclear localization signal (NLS) and is largely cytoplasmic, co-expression with HCF resulted in VP16 nuclear accumulation. A candidate NLS within the C-terminus of HCF was identified and insertion of this motif into green fluorescent protein (GFP) promoted nuclear accumulation. Conversely, removal of this signal from HCF (HCFDeltaNLS) resulted in its cytoplasmic accumulation. Co-expression of HCFDeltaNLS with wild-type (wt) VP16, or of wt HCF with VP16 mutants lacking HCF-binding activity failed to promote the nuclear enrichment of VP16. These results indicate that in addition to its role in stabilizing TRF.C, HCF acts as a nuclear import factor for VP16.