Dose Of Cy Research Articles

HLA-Matched Sibling Stem Cell Transplantation Using Conditioning Regimen with Reduced Dose of Cyclophosphamide, ATG and Fludarabine in Patients with Severe Aplastic Anemia

Background: We have reported the outcome of HLA-matched stem cell transplantation (SCT) using triple immunosuppressive agents with cyclophosphamide (CY, 200mg/kg), ATG and procarbazine for 113 adult patients with severe aplastic anemia (SAA) (Kim et al, BMT 31:79, 2003). However, high dose CY (200mg/kg) causes serious cardiac toxicity in some cases which may lead to death within few weeks. To avoid high dose CY-associated cardiac toxicity we underwent HLA-matched sibling SCT using increasing dose of ATG and Fludarabine instead of reduced CY to half dose.Method: Between March 2002 and December 2007, fifty patients with adult SAA (six patients were AA/PNH syndrome) received matched sibling SCT. The median age of patients was 39 (16~53) and median interval between Dx and SCT was 19 months (1~352). The median number of transfusion prior to SCT was 34 units (2~680). Nineteen patients (38%) had a history of IST before SCT. The conditioning regimen consisted of Fludarabine (30mg/m2/day, 6 days), CY (50 mg/kg/day, 2 days) and ATG (Thymoglobulin 2.5mg/kg/day, 4 days). Stem cell sources were BM plus CD34+-selected PBSC (n=12), BM (n=20), PBSC (n=4) and G-CSF-primed BM (n=14). All patients received cyclosporine and methotrexate as GVHD prophylaxis.Results: The median dose of CD34+ cells infused was 3.7x106/kg (1.2~11.9). All patients achieved successful primary engraftment, and the median time for ANC and platelet to reach 0.5x109/L and 20x109/L was 12 (10~19) and 17 (10~25) days, respectively. Three patients (6%) developed delayed graft failure whereas 14% (16 out of 113) developed both primary and secondary graft failure in our previous study. But all achieved successful engraftment after booster infusion (n=1) and second SCT (n=2). The incidence of acute GVHD (more than grade II) and chronic GVHD was 8% (n=4) and 4% (n=2; extensive type), respectively. The incidence of acute and chronic GVHD seems to be lower than those of previous conditioning regimen (11% and 12%, respectively) (BMT 31:79, 2003). The incidence of CMV infection requiring preemptive treatment was 54 % (n=27). Three patients died of reactivation of chronic hepatitis C with hepatic GVHD (n=1), CMV pneumonia (n=1), and invasive fungal infection (n=1). PNH clone monitored by flow cytometry disappeared posttransplant in all 6 PNH patients. With median follow up of 32 months (1~74), the estimated probability of survival at 3 years was 94 % compared with those of 89% in our previous report.Conclusions: These data demonstrate that the conditioning regimen used in this study is feasible for patients with SAA who received matched sibling SCT. Of note, the observation of successful engraftment as well as lesser acute and chronic GVHD compared with previous study suggest that increasing dose of ATG and the addition of Fludarabine has potent in vivo T cell depletion and immunomodulatory activity.

Post-Transplantation High Dose Cyclophosphamide (Cy) Is Effective Single Agent for Prevention of Acute and Chronic Graft Versus Host Disease after Myeloablative HLA Matched Related and Unrelated Bone Marrow Transplantation (BMT)

Based on our results in animal models and promising results in HLA-haploidentical setting (L. Luznik, Blood 2002 and BBMT 2008), we studied whether high dose Cy alone is sufficient as GVHD prophylaxis after myeloablative HLA-matched related or unrelated BMT in patients with advanced, poor risk, hematologic malignancies. One hundred and seventeen consecutive patients (median age 48, range 23–65; 36 de novo AML, 23 2° AML (arising from previous MDS/MPD or therapy-related), 13 high-risk MDS, 9 ALL, 8 CML, 3 CLL, 5 MM, 9 NHL and 11 HL), of whom the majority (57%) were not in remission, received HLA-matched related (n=78) or unrelated (n=39) BMT. Conditioning consisted of oral or IV busulfan (pharmacokinetically adjusted) on days −7 to −3 and Cy (50 mg/kg/day) on days −2 and −1. Cy (50 mg/kg/day) was also given on days +3, and +4 as a sole agent for GVHD prophylaxis. All patients received bone marrow allografts without growth factor support. Three patients failed to engraft, but two were successfully rescued with a second allograft. The cumulative incidence of non-relapse mortality (NRM) at day 100 and 1 year after transplantation was 8.5% and 16%, respectively. Of the 18 patients dying of NRM, 2 were from VOD, 3 from non infectious pneumonia, 3 were from GVHD, 3 from sepsis/bacterial infections, 4 from MOF, and 3 of CNS/organ hemorrhage. The incidences of acute grade II–IV and grade III–IV GVHD were 43% and 11%, respectively. With a median follow-up of 19 months, 66 (56.4%) patients are alive, of whom 52 (44.4%) are in complete remission. Only 7/66 related and 4/32 unrelated patients developed chronic GVHD (classic limited in 7, overlap syndrome limited in 1, and classic extensive in 3 patients). Since in a competing risk model (relapse and death as competing risks) the cumulative incidence of chronic GVHD remained low, we analyzed the impact of the preceding history of acute GVHD and systemic immunosuppressive treatment given beyond the originally prescribed prophylaxis with high dose Cy on the incidence of chronic GVHD. Only one patient without a preceding history of acute GVHD developed de novo chronic GVHD. Overall, 3 patients with grade II–IV acute GVHD were untreated, 10 patients received steroids alone, 31 received steroids + a calcineurin inhibitor (CNI), and 7 received steroids + non CNI-based agents. The use of CNI for the treatment of acute GVHD did not appear to influence the development of chronic GVHD: 6/31 (19%) patients who received CNI-based immunosuppressive treatment developed chronic GVHD compared to 3/20 (15%) patients who did not. These results suggest that highdose of post-transplantation Cy is effective as the sole prophylaxis for acute and chronic GVHD after HLA-matched related or unrelated BMT. This approach is associated with rapid immunologic recovery as indicated by the low incidence of opportunistic infections, as well as a low incidence of acute and especially chronic GVHD. Further clinical and correlative studies are needed to elucidate the mechanisms behind the unique effectiveness of post-transplantation Cy on the prevention of acute and chronic GVHD.

Autologous Peripheral Blood Stem Cell Products from Patients with Hematologic Malignancies Have Increased Frequency of Regulatory T Cells (CD4+FoxP3+ Treg).

Increased numbers of regulatory T cells (Treg) with immune suppressive function have been observed in patients with hematological malignancies. Many of these patients will undergo stem cell mobilization with high dose cyclophosphamide and G-CSF to prepare for autologous stem cell transplantation. Although infusion of low dose cyclophosphamide (Cy) seems to reduce the number of circulating Tregs, mobilization with high dose Cy and G-CSF was shown to increase the frequency of immunosuppresive Treg in stem cell autografts of myeloma patients. Treg populations have been defined by different phenotypic criteria, including CD4+FoxP3+ and CD4+CD25+ CD127lo. In this study, we compare the impact of donor types and stem cell mobilization regimens on CD4+FoxP3+ and CD4+CD25+ CD127lo Treg frequencies in 63 apheresis products.Methods: Apheresis products from 23 healthy donors (17 donors received G-CSF and 6 had no stem cell mobilization) and 40 patients with hematological malignancies (1 AML, 2 HD, 25 NHL, 10 MM, 2 neuroblastoma) were evaluated. Patients with malignancies were mobilized with chemotherapy (5 RICE, 25 Cy, 1 Ara-c+Etoposide) + G-CSF (N=31), AMD3100 + G-CSF (N=5), or G-CSF alone (N=2). Samples from each product were incubated with fluorochrome-conjugated antibodies against cell surface makers, CD45, CD4, CD14, CD127, and CD25, followed by intracellular staining with anti-FoxP3 antibody. Six-color flow cytometry and sequential gating was performed for each sample to determine the frequency of CD4+Foxp3+ and CD4+CD25+CD127lo Treg population.Results: A strong correlation was found between CD4+Foxp3+ and CD4+CD25+CD127lo Treg subsets (r=0.67, p<0.001).The percentage of both Treg populations was not different between mobilized or non-mobilized products from healthy donors (p=0.94) or between NHL and MM patients (p=0.81). Higher CD4+FoxP3+ Treg frequency was observed in autologous grafts from patients compared to healthy donors (p<0.001). The median CD4+FoxP3+ Treg frequency among mobilized products from patients and healthy donors were 8.5% (range, 1.8–38.9) and 4.3% (range, 1.8–15.0), respectively. Autografts from patients who received AMD3100+G-CSF compared to those who received chemotherapy + G-CSF also revealed higher CD4+FoxP3+ Treg content (median 11.3 vs. 7.5, p=0.04). Similar analysis using CD4+CD25+CD127lo Treg subset did not demonstrate any statistical significance.Conclusions: Although correlation was observed between CD4+FoxP3+ and CD4+CD25+CD127lo subsets, CD4+FoxP3+ is a more sensitive marker for the Treg population in stem cell products. Higher CD4+FoxP3+ Treg frequency was observed in grafts from patients with hematological malignancies compared to healthy donors. The addition of a novel mobilization agent, AMD3100, to Cy mobilization may further increase the Treg content of autologous stem cell products.

Combination chemo-immunotherapy: kinetics of in vivo and in vitro generation of natural killer cells and lymphokine-activated killer cells in the rat

Rats received a single high dose of cyclophosphamide (Cy) (150 mg/kg), followed 48 h later (on day 0) by immunization with a T cell-dependent soluble antigen, ovalbumin in Freund's complete adjuvant (FCA). The effect of this treatment on lymphoid cell subpopulations in the spleen, natural killer (NK) cell and interleukin-2 (IL-2) induced lymphokine-activated killer (LAK) cell activity was examined. Cy (with and without ovalbumin) caused a large relative increase (by day 14) in splenic OX8+, OX19- cells with NK morphology. A marked relative increase in fresh NK cell activity was noted after Cy + ovalbumin, but not consistently after Cy alone. Elevated NK activity was Cy dose- and time-dependent, was evident within 7 days post Cy/ovalbumin and persisted for at least 28 days. Pooled splenic mononuclear cells (MNC), obtained 14 days after Cy/ovalbumin, lost all cytolytic activity against YAC-1 cells when cultured in the absence of human recombinant IL-2 (rIL-2). In contrast, similarly maintained cells from normal rats displayed NK activity higher than normal 'fresh' levels. Upon culture in medium containing 500 U/ml rIL-2, however, 'augmented' NK activity was equivalent, on a per-cell basis, in both normal and Cy/ovalbumin-pretreated groups. LAK activity generated in vitro (i.e. against NK-resistant target cells) was significantly lower in the latter group, and the overall yield of cells was reduced. By day 21 after Cy/ovalbumin, augmented NK activity was significantly greater than controls, on a per-cell and total culture yield basis. Moreover, LAK activity was now similar between groups. It is concluded that the chemotherapy/immunization protocol which we have used can greatly enhance NK activity in vivo and that these cells are responsive to induction of LAK activity by IL-2 in vitro.

Reduced Intensity Conditioning (RIC) with Fludarabine, Busulfan and Cyclophosphamide for High Risk Patients with Thalassemia Major Undergoing Allogeneic Bone Marrow Transplantation Results in High Rejection Rates.

Usual cytoreductive therapy for bone marrow transplantation (BMT) for thalassemia major consists of busulfan (Bu) 14–16mg/kg and cyclophosphamide(Cy) 160–200mg/kg +/− anti-thymocyte globulin (ATG). In high risk patients, this is associated with significant regimen related toxicity and graft rejection. Fludarabine (Flu) containing conditioning has been used effectively by adding immunosuppression in RIC with reduced doses of myelosuppressive drugs to lower regimen related toxicities and graft rejection. There is also data to suggest that a 24-hour gap between Bu and Cy doses could reduce toxicities associated with this combination. We therefore treated 25 patients (median age: 7 years, range: 3–14), 12 males and 13 females, with beta thalassemia major with a regime of Flu 150mg/m2 (day-15 to -11), Bu 14mg/kg (day-10 to -7) and Cy 160mg/kg (day-5 to day-2). Most of these patients were heavily transfused (median red cell units:100, range: 21–250) and poorly chelated (median serum ferritin: 2510 ng/ml, range: 1039–6740). Their risk stratification (Lucarelli class) was as follows: Class I: 4 (16%), Class II: 8 (32%) Class III: 13 (52%). Allogeneic BMT was performed using a 6/6 matched related donor and bone marrow as the graft. Graft versus host disease (GVHD) prophylaxis consisted of cyclosporine A and mini-methotrexate. The median cell dose was 4.56 × 108 TNC/kg (range: 2.5–14.1). Three (20%) of 20 assessable patients developed grade I-II acute GVHD, 2 (10%) each had hemorrhagic cystitis and veno-occlusive disease. Mortality within the first 100 days were due to graft failure/rejection in 4 patients (16%) and one each of diffuse alveolar hemorrhage, intracranial hemorrhage and sepsis (4% each). With a mean follow-up of 8 months (range 0–11), 18 (72%) of 25 patients are alive. Among the 13 patients in class III, 5 (38.5%) rejected the graft (3 had primary failure and 2 after initial engraftment). In a historical cohort of 47 patients conditioned with Bu16, Cy200 and ATG, the rejection rate was only 8%. The regimen was very well tolerated by all patients and patients in class I had no rejection. Pharmacokinetic data was available on Bu and Cy from 14 of the 25 patients treated with this protocol. The mean values for Bu kinetics were: Cmax-1 (ng/ml): 1069.2±265.8, Cmin-1(ng/ml): 186.8±73.2, Cl-F-1 (l/h/kg): 0.281±0.081, AUC-1 (ng*h/ml): 3648±918 and Css-1 (ng/ml): 608±153. They are not significantly different from patients conditioned Bu16mg/kg without Flu (data not shown). The Cy kinetic data are as follows: Cmax-1(ng/ml): 441.4±188, AUC-1(ng*h/ml): 1431±791, Cl-F-1(l/h/kg): 0.0378±0.027. The Cmax-1 and AUC-1 were significantly lower compared to patients receiving 200mg/kg and dosed without a 24-hour gap between the Bu and Cy. Overall, these data suggest that Flu does not provide adequate immunosuppression for sustained engraftment after allogeneic BMT even when combined with Bu 14 and Cy160 (with a 24-hour gap between Bu and Cy) in high risk patients with thalassemia major (Lucarelli class III) but could be useful in the others (Class I and II).OUTCOME OF ALLOGENEIC BMTCLASSNUMBER (%)OVER ALL SURVIVAL (months)EVENT FREE SURVIVAL (months)REJECTION (%)MORTALITY (%)ALL PATIENTS2569±9.862.6±10.06 (24)7 (28)CLASS I4 (16%)75±21.775±21.701 (25)CLASS II8 (32%)83.3±15.283.3±15.21 (12.5)1 (12.5)CLASS III13 (52%)57.7±14.744.9±14.15(38.5)5 (38.5)

A New Mice Model of Graft Versus Host Disease Based on Chemotherapy Conditioning.

Background: Transplantation related complications such as graft versus host disease (GVHD) are limiting factors for allogeneic haematopoietic stem cell transplantation (HSCT). The majority of mice models of GVHD are based on the use of radiotherapy as a conditioning regimen. However, these models may not explain mechanisms of HSCT related complications following chemotherapy-based conditioning regimen.Aims: The aim of this study was establishment of a mice model of GVHD using the chemotherapy as conditioning regimen.Methods: Recipient female BALB/c (H-2Kd) were conditioned using busulfan (Bu) in total dose of 80 or 100 mg/kg followed by cyclophosphamide (Cy) in total dose of 200 or 300 mg/kg. Twenty millions of bone marrow (BM) cells and 30×106 spleen cells of male C57BL/6 (H-2Kb) were injected into the recipient mice in allogeneic setting. As a control, syngeneic HSCT using female BALB/c as recipients and donors and the same conditioning regimen and cell doses as in allogeneic setting was used. Acute GVHD manifestations and histo-pathological changes in skin, liver and intestine were evaluated at different time points after BMT. The chimerism and tissue mapping were studied by FACS, immunohistochemistry and FISH analysis. Affected skin was examined by FACS analysis to find donor T cells. Cytokines level were measured by using luminex assay.Results: Infusion of 20×106 BM cells and 30×106 spleen cells from C57BL/6 mice to BALB/c mice conditioned with 80 or 100mg/kg Bu plus 200mg/kg Cy induced lethal acute GVHD between days 5–7 post transplantation. Histological and clinical signs of GVHD were found in 85% and 100% of the allogeneic transplanted mice based on the intensity of conditioning. About 35% of mice in allogeneic group died due to GVHD progression. The number of donor T cell in the recipient skin increased with GVHD progression and was correlated to the level of chimerism in the spleen but not in the BM. CD3+ CD8+ T cells infiltration and apoptosis were found in the skin, intestine and liver of mice suffering from GVHD. None of the syngeneic transplanted mice died or developed any sign of GVHD even in long term follow up, and CD3+CD8+ T cells were absent in their tissue samples. Increasing the dose of Cy in conditioning regimen in allogeneic transplanted mice increased mortality of animals.Conclusion: We have established a new mouse model of acute GVHD using chemotherapy based conditioning. This model can be utilized to study the basic mechanisms underlying GVHD triggered by cytostatics used in the conditioning regimen and affecting different cell sub-populations and cytokine storm. [Display omitted] [Display omitted]

High Dose Sequential (HDS) Followed by Autologous Hematopoietic Stem Cell Transplantation (AHSCT) for Salvage Treatment of Hodgkin's Disease (HD): A Brazilian Experience.

From May 1998 to November 2006, 77 HD patients who used the regimen of high dose cyclophosphamide HD (CY) 7 or 4g/m2, methotretaxe 8g/m2 and etoposide 2g/m2 followed by AHSCT were analyzed. Their median age was 25.8 (8.8–71.5) years, 46 males and 31 females. At diagnosis 50 (65%) were stage III or IV disease, 10 (13%) had bone marrow involvement, 29 (37.7%) bulky disease and 55 (71.4%) B symptoms. Besides that, all patients were submitted to a mean of 2 (1–4) chemotherapy lives and their status were 3 (3.9%) complete remission (CR), 17 (22.1%) partial remission (PR), 57 (74%) progression disease (PD) or in non-specified sensitivity relapse. Concerning CY dose 30 (39%) received 4 g/m2 and 47 (61%) received 7 g/m2. After the HDCY 16 (20.8%) were in CR, 22 (28.6%) PR, 28 (36.3%) remained in DP and 11 (14.3%) died most in PD. After a median follow-up of 3.97 (1.13–55.9) months, 53 (68.8%) patients were submitted to AHSCT and their present status is 30 alive [18 CR, 2PR and 10 DP]; 23 dead [2 CR, 14PD and 7 related to the procedure]. Overall survival for transplanted patients was 55% in 8 years. Currently we have 33/77 (43%) alive patients, 19 CR, 3 PR, 11 PD. Overall Survival (OS) for whole group was 32%, Disease Free Survival (DFS) 64% and Progression Free Survival (PFS) 40%. Patients who were in DP or relapse prior to the HDCY (57) compared to their status after that had a significant improvement (P=0,001), their OS was 40% to CR-PR group (24) versus 16% PD group (33). In general, mortality was 44 (57%), their cause was 19 PD (43.2%), 8 (18.2%) related to HDCY, 2 (4.5%) related to HDMTX, 7 (16%) related to AHSCT, 6 (13.6%) infections, 1 (2.3%) chronic GVHD after a reduced intensity conditioning regimen transplant and 1 (2.2%) AML. Besides that, 3 patients developed MDS and 1 developed AML. In conclusion, although it had happened a significant number of toxicity related deaths, the sequence is feasible, mainly for sensitive patients and presents an acceptable response. The authors emphasize the high frequency of poor prognosis patients and occurrence of MDS/AML in 4 (5.2%) patients.

Brazilian Experience Using High Dose Sequential (HDS) Followed by Autologous Hematopoietic Stem Cell Transplantation (AHSCT) for Relapse/Refractory Aggressive Non-Hodgkin Lymphoma (NHL).

The purpose of this study was to evoluate the use of high dose cyclophosphamide HD (CY) 7 or 4g/m2 and HD etoposide (VP-16) 2g/m2 followed by AHSCT in patients affected by aggressive NHL. From January 1998 to November 2006, 106 patients, 66 males, 40 females, median age 47 (8–66) years underwent into this regimen. The diagnosis was 83 (78.3%) DLBCL, 13 (12.3%) with T-cell Lymphoma and 10 (9.4%) Mantle Cell. At diagnosis, 88 (83%) were stage III or IV, 34 (32.1%) presented bone marrow involvement, 65 (61.3%) bulky disease, 67 (63.2%) B symptoms and 45 (42.5%) were high or high intermediate risk according to IPI. Prior to HDCY patients had been submitted to a mean of 2 (1–3) therapy lines and 6 (5.7%) were in complete remission (CR), 38 (35.8%) partial remission (PR), 62 (58.5%) disease progression (PD) or relapse. Concerning CY dose 42 (39.6%) patients received 4 g/m2 and 64 (60.4%) 7 g/m2, respectively. After HDS, 13 (31%) patients who had used 4 g/m2 and 30 (47%) used 7 g/m2 were in CR (P= 0.03). After the HDCY 43 (40.6%) were in CR, 33 (31.1%) PR, 21 (19.8%) remained in PD and 9 (8.5%) died. After a median follow-up of 4.1 (1.5–57) months, 80 patients (75.5%) were submitted to AHSCT and their present status is 44 alive [33 CR, 4 PR, 7 PD]; 36 dead [4 CR, 1 PR, 2 relapse, 17 PD and 12 had death related to procedure]. Their overall survival was 45% in 8 years. Currently we have 50/106 (47%) alive patients, 35 CR, 6 PR and 9 PD. Overall Survival (OS) was 37%, Disease Free Survival (DFS) 49%, Progression Free Survival (PFS) 42%, Event Free Survival (EFS) 28%. OS by diagnosis was 42% DLBCL, 40% T-cell in 8 years whereas 20% Mantle Cell in 6 years (P=NS). OS by B symptoms patients was 22% versus 58% (P= 0.002) and EFS was 23% versus 37% (P= 0.03). Patients who were in PD prior to the HDCY compared to their status after that had a significant improvement (P< 0.001), their OS was 38% to CR-PR group (38) versus 0% PD group (24). In general, mortality was 56 (53%), their cause was 23 (41.1%) PD, 7 (12.5%) related to HDCY, 12 (21.4%) related to AHSCT, 13 (23.2%) infections and 1 (1.8%) GVHD a RIC transplant. Besides that 1 (0.9%) patient developed MDS and is alive. B symptoms at diagnosis have appeared as a predictor factor for survival. Our study suggests HDS is an efficient treatment to improve status and to reduce tumor burden. Although it presented high toxicity related mortality, we consider the treatment feasible, especially considering the patients' poor prognosis.

A novel xenograft model to test the ovarian toxicity of chemotherapy agents in women

19504 Background: Many chemotherapeutic agents, especially of alkylating family, alter fertility in premenopausal females (1). While the impact of well-established agents such as cyclophosphamide (Cy) on fertility is well known (2), data are not available for newer agents. Moreover it is not practically possible to determine the individual ovarian-toxicity of each drug and the cellular mechanism of this damage from clinical trials.Thus our specific aim was to develop a xenograft model to characterize the impact of chemotherapy agents on human ovary. Methods: Ovarian pieces of 2x2 mm from 24 wk old abortuses were xenografted subcutaneously to immunodeficient mice (SCID) (n=52). At this gestational age ovarian histology is simiar to young females. Half of the animals received a single dose of 200 mg/kg of Cy intraperitoneally 14 days after the xenografting, while the other half received saline. Grafts were recovered from the control and treated mice 12, 24, 48, and 72h after the Cy injection (n=6–7 per time point), and serially sectioned for primordial follicle counts. Apoptosis was assessed with TUNEL assay. PECAM-1 staining, as well as intra-vital fluorescein conjugated lectin and Evans blue labeling were performed to assess microvasculature by confocal microscopy. All experiments were performed in triplicate. Results: While there was only 12 % loss 12h following the treatment compared to controls (p&gt;0.05), the follicle loss significantly increased at 24h (53%, p&lt;0.01) and peaked at 48h (93 %, p&lt;0.0001), and reduced at 72h (70 %, p&lt;0.001). TUNEL staining peaked at 12h, earlier than the follicle loss; then decreased at 24h and 48h, and faded at 72h. Less than 1% of follicles were TUNEL positive in controls. Xenograft vasculature was similar to non-xenografted tissue indicating that the ex vivo tissue drug delivery was similar to the in vivo delivery. Conclusions: A single injection of a typical dose of Cy can result in the loss of up to 93 % of ovarian reserve in a human ovarian xenograft model, consistent with the data from clinical trials. Fetal ovarian xenografting is a promising model to characterize the gonadotoxic effects of current and emerging cancer drugs without a need for lengthy clinical studies. 1. J Clin Oncol. 2006;24:2917–31 2. Hum Reprod Update. 2004;10:251–66. No significant financial relationships to disclose.

Enhancement of Pavlovian conditioned immunosuppression in rats

The goal of this study was to define conditions under which conditioned immunosuppression may be observed reliably. In three experiments, rats were exposed to a gustatory conditioned stimulus (CS) paired with cyclophosphamide (US), which induces immunosuppression and malaise. In Experiment 1, a single pairing of the CS with low, medium, or high doses of cyclophosphamide in separate groups produced no reliable conditioned immunosuppression even though conditioned taste aversion was observed in groups trained with high and medium doses of CY. Experiment 2 replicated the lack of effect following a single pairing of the CS with the medium dose of cyclophosphamide but demonstrated that three pairings are sufficient to induce conditioned immunosuppression. Experiment 3 demonstrated that significant immunosuppression is observable following a single CS-US pairing if the CS is presented in compound with a previously nonreinforced CS during training, an effect reminiscent of supernormal conditioning. These findings indicate that conditioned immunosuppression effects can be enhanced in magnitude through the use of certain procedural techniques. Correspondence should be addressed to E. Vogel, Email: evogel@utalca.cl

Busulphan in Blood and Marrow Transplantation: Dose, Route, Frequency and Role of Therapeutic Drug Monitoring

Busulphan (Bu) is an alkylating agent that, when combined with agents such as cyclophosphamide (Cy), ablates the bone marrow prior to blood or marrow transplantation. There is wide inter- and intra- patient variability in Bu pharmacokinetics. Early pharmacodynamic studies suggested a significant relationship between high Bu exposure and the occurrence of veno-occlusive disease of the liver, but were not performed in uniform patient populations and tended to use a Cy dose of 200 mg/kg. Pharmacodynamic studies in uniform patient populations, with lower doses of Cy, contradict these results. Despite 20 years of clinical use, pharmacodynamic studies are still required to define the relationship between Bu exposure and optimal transplant outcome, and these may vary according to age, disease, transplant type and conditioning regimen. Given the availability now of an intravenous formulation, it is timely to review how and why we are giving Bu. Administration of single daily doses of Bu has been shown to be safe and effective with oral Bu and has been used with i.v. preparations. This simple administration provides accuracy in measuring exposure, which is ideal for pharmacokinetic studies, and provides the possibility of defining a target exposure level associated with a good outcome. Only when this target is defined will therapeutic drug monitoring be useful to minimise toxicity, maximise efficacy and improve transplant outcome.

Determination of the Maximum Tolerated Dose of Cyclophosphamide in Conjunction with High-Dose Etoposide and Carboplatin Followed by Autologous CD34+ Hematopoietic Rescue in Pediatric Patients with Recurrent/Refractory Solid Tumors.

Cyclophosphamide (Cy) is an alkylating agent utilized in almost all preparative regimens for hematopoietic stem cell transplantation, and in vitro sensitivity has been demonstrated with carboplatin and etoposide. Multiple previous studies have involved this combination of agents; however, they almost exclusively utilized a fixed dose of Cy and/or bolus administration. The potential fatal cardiac toxicity of Cy is likely related to high peak concentrations of active metabolites from bolus dosing. Additionally, some evidence exists that the increased metabolism of Cy with continuous infusion has a therapeutic benefit. The objective of this study was to determine the maximum tolerated dose (MTD) of continuous infusion Cy with fixed dose etoposide (2400 mg/m2) and carboplatin (2175 mg/m2) followed by autologous CD34+ hematopoietic rescue in pediatric patients with recurrent/refractory solid tumors. Twenty patients were enrolled, and fourteen patients completed therapy on study. The MTD of Cy was 60 mg/kg/24 hr by continuous 60 hr infusion on days -4 to -2 for a total dose of 150 mg/kg. Alopecia, stomatitis and nausea were the most common toxicities. The single episode of direct cardiac toxicity was reversible. Two Grade IV toxicities occurred, consisting of diarrhea in one patient and coma in another patient who recovered and remains a long-term survivor. All patients were evaluable for neutrophil engraftment, with a median time to ANC > 200/μl of 11 days (range 7–30 days). Thirteen of the fourteen patients were evaluable for platelet engraftment, with a median time to platelet count of 20,000/μl independent of transfusion of 21 days (range 11–72 days). Six out of the fourteen patients remain alive greater than five years after study completion. The MTD of Cy was lower than expected. These results suggest that autoinduction of Cy metabolism (repeated administration of Cy at 24 hour intervals leads to increased hepatic cytochrome enzymes) could be a primary factor in the determination of the MTD. Future studies should aim to further characterize the pharmacokinetics and effectiveness of continuous infusion Cy in pediatric patients.

Impact of Killer Immunoglobulin Receptor (KIR) Ligand Incompatibility in Nonmyeloablative Bone Marrow Transplantation (BMT) from Haploidentical Donors.

Donor vs. recipient natural killer (NK) cell alloreactivity in mismatched (haploidentical) bone marrow transplantation (BMT) has been defined as a mismatch between killer inhibitory receptors (KIR) on donor NK cells and their inhibitory HLA class I ligands on recipient cells. Data reporting the impact of KIR incompatibility in the haploidentical BMT setting have been controversial. Here, we examine the effect of KIR ligand mismatches in 60 patients with advanced hematologic malignancies who were enrolled in a clinical trial of nonmyeloablative haploidentical BMT (“mini-haploBMT”). All patients were conditioned prior to transplantation with fludarabine, low dose cyclophosphamide (Cy), and 200 cGy TBI, and after transplantation received high dose Cy, mycophenolate mofetil (MMF) and tacrolimus. The median age of transplant recipients was 48 years (range, 1–71years). Patients received a median of 1.27x 108/kg mononuclear cells/kg and a median of 3.9 x 106 CD34+ cells/kg. Donor-recipient pairs had an average of 3 out of 8 HLA antigen mismatches in both the host-vs.-graft (HVG) and graft-vs.-host (GVH) directions. Graft failure occurred in 12/58 (21%) evaluable patients, and grade 3–4 GVHD occurred in 7/60 patients (11.7%). Median time to disease progression was 222 days and median overall survival was 255 days. The median follow-up at the time of this analysis was 251 days (range 41–1715 days). KIR mismatches were identified via KIR genotyping (KIR genes determined by SSP from genomic DNA extracts) as well as via the previously described receptor:ligand model which identifies the presence of inhibitory KIR on donor cells, with absence of HLA ligand on host cells. Utilizing the receptor:ligand model, our results demonstrate an improvement in time to relapse (TTR) in patients with ≥1 inhibitory KIR mismatches (32 patients, median TTR 359 days) versus no mismatches (27 patients, median TTR 185 days) (p=0.036) and an improved overall survival (OS) in patients with ≥1 inhibitory KIR mismatches (32 patients, median OS 622 days) versus no mismatches (27 patients, median OS 208 days) (p=0.008). No significant difference in OS or TTR was found when patients were separated by disease type (myeloid versus lymphoid; OS p= 0.095 vs. p=0.1 and TTR p=0.2 vs. 0.1). There were also no significant differences in TTR or OS when using the KIR genotyping method of identifying mismatches. No differences in Grades 2–4 graft-versus-host disease (GVHD) or engraftment were found based on the presence of inhibitory or stimulatory KIR mismatches, although the small numbers of patients with GVHD or engraftment failure prevent a definitive analysis. KIR ligand incompatibility in the GVH direction is associated with a prolonged time to relapse and improved overall survival after nonmyeloablative haploidentical BMT. These findings may warrant the selection of donors based upon KIR ligand incompatibility.

O-223: A novel ovarian xenografting model to characterize the impact of chemotherapy agents on human primordial follicle reserve

Cyclophosphamide (Cy) is the most commonly used chemotherapy agent and it has a significant negative impact on fertility. While it is believed that Cy results in primordial follicle loss through an apoptotic process, the exact mechanism and the time course of this action is unknown in humans. Our specific aim was to characterize the impact of the usual dose of Cy on human primordial follicle reserve while developing a xenograft model to determine the gonadotoxicity of chemotherapeutics.

Vinorelbine plus intermediate dose cyclophosphamide is an effective and safe regimen for the mobilization of peripheral blood stem cells in patients with multiple myeloma

High dose cyclophosphamide (HD-Cy) is commonly used to mobilize stem cells in multiple myeloma (MM). However, timing of collection is variable and incidence of side effects is substantial. We evaluated a combination of vinorelbine (VNB) (25 mg/m(2) day 1) plus Cy (1.5 g/m(2) day 2) and G-CSF as mobilizing regimen in 37 patients with MM. Results were compared to those achieved in 41 previously diagnosed patients mobilized with Cy at 4 g/m(2). Overall, 36/37 patients receiving VNB-Cy (97%) mobilized, as opposed to 40/41 (97%) in the controls (p:0.51). Median CD34+ cells peak was 94/mul for VNB-Cy patients and 96 for controls, p=0.36; median number of CD34+ cells collected was 9.2x10(6)/kg and 8.7x10(6)/kg, respectively (p=0.85). Median number of days to the highest CD34 count was shorter for VNB-Cy patients (nine vs 11, p=0.001). No VNB-Cy patient experienced grade 3-4 neutropenia and thrombocytopenia, as opposed to 63 and 19% in the controls (p=0.001 and 0.01, respectively). Hospitalization from toxicity was never required in VNB-Cy patients as compared to 19% in control group (p=0.01). We conclude that an outpatient combination of VNB plus intermediate dose Cy plus G-CSF is a safe, predictable, and highly effective mobilization regimen for patients with newly-diagnosed MM.

Stem cell transplantation in 40 pts with Fanconi anemia (FA): Excellent survival and low toxicity for pts with a related HLA identical donor

SCT is the treatment of choice for FA patients (pts). These pts are particularly hypersensitive to alkylating agents and radiation. In order to decrease toxicity and maintain engraftment we have progressively decreased the dose of CY during the last 20 years. In this study we report our experience using the dose of CY 60 mg/kg in 40 pts transplanted between 07/99–04/05. Median age : 9 years (5–29). Previous blood transfusions: 0–101 (M: 5UI). Previous treatment with androgens: 15/40 pts. All pts had pancytopenia and received either unmanipulated bone marrow (39 pts) or cord blood (1 pt) from HLA identical donors (siblings: 33 pts, other related: 7 pts).

Low Incidence of CMV Reactivation and Infectious Morbidity and Mortality after Nonmyeloablative Haploidentical Bone Marrow Transplantation Incorporating Post-Transplantation Cyclophosphamide.

Graft rejection, severe graft-versus-host disease (GVHD), and prolonged immunosuppression are major complications of partially HLA-mismatched, or haploidentical, stem cell transplantation in humans. Intensive conditioning of the recipient and rigorous T cell depletion of the donor graft reduces the risk of the first two complications, but may prolong or intensify post-transplantation immunodeficiency, increasing the patient's susceptibility to opportunistic infection. In contrast, alloreactive T cells may be selectively eliminated in vivo by the early post-transplantation administration of high dose cyclophosphamide (Cy), leaving virus or other pathogen specific resting memory T cells relatively unharmed. To test this hypothesis, we have analyzed the infectious complications in 60 patients who were enrolled in a clinical trial of nonmyeloablative haploidentical bone marrow transplantation (“mini-haploBMT”) incorporating post-transplantation Cy. All patients received fludarabine 30mg/m2/dose on days -6 to -2, Cy on day -2, and 200 cGy total body irradiation (TBI) on day -1. The first 20 patients received a single dose of 50 mg/kg Cy IV on day 3, whereas the final 40 patients received the same dose of Cy on day 3 and 4. The median age of transplant recipients was 48years (1-71years), 12 patients had 13 prior autologous transplants prior to enrollment, and patients had an average of 2.7 treatment regimens prior to transplant (0–8). Patients received a median of 1.27x 108/kg mononuclear cells/kg with 3.9 x 106 CD34+ cells/kg. The median follow-up at the time of this analysis was 245.5 days (41–1550 days). An ANC >500/mm3 was achieved a median of day 16 (12–208). Only 5 patients (8.3%) died from infectious related causes. Of these, 3 were known to have relapsed disease. There were 34 patient/donor pairs (56.6%) at risk for CMV reactivation. In these pairs, only 6 patients developed CMV reactivation (17.6%), and 2 patients developed CMV infection. 3 patients with CMV reactivation received one dose of post-transplant Cy, and 3 patients received two. Both patients who developed CMV infections received only dose of post transplant Cy. 23% (14/60) of patients developed an invasive fungal infection, of which 21.4% (3/14) developed in the first 30 days post transplant, 14.3% (2/14) developed between days 31–60, 14.3% (2/60) developed between days 61–100, and 50% (7/14) occurred more then 100 days post transplant. 15% (9/60) had at least one systemic viral infection (other then CMV), of which 22.2% (2/9) developed in the first 30 days post-transplant and 77.8% (7/9) developed more then 90 days post transplant. No patients developed viral infections between days 31–100. These rates of infection compare favorably to the rates of infection seen in patients receiving haploidentical grafts following myeloablative conditioning without post-transplantation Cy. We propose that the very low numbers of patients with CMV reactivation, fungal or viral infections, and infectious related mortality is secondary to early engraftment, low rates of Grade 3–4 GVHD (7/60 patients, 11.7%; 5/7 patients received one dose of post-transplant Cy, 2 received two) or prolonged immunosuppression, and an intact host B cell and memory T cell function preserved with this novel prep regimen and T cell replete graft.

Successful Autologous Stem Cell Transplantation in Patients with Non-Hodgkin's Lymphoma over the Age of 70 Years.

Autologous stem cell transplantation (SCT) is a potentially curative procedure for patients with relapsed lymphoma (NHL). Over 60% of the cases arise in adults over 60 years of age. We retrospectively analyzed the outcomes of patients >60 years old who received high dose busulfan (Bu) and cyclophosphamide (Cy) and autologous SCT for NHL. Stem cells were mobilized with G-CSF with or without chemotherapy. Patients received a dose-adjusted regimen of Bu 0.8 mg/kg IV (n=17) or 1mg/kg po (n=9) q 6 hours x 14 doses starting on Day -7 and Cy 60 mg/kg IV q day on days -3 and -2. In overweight patients, IV Bu doses were calculated based on ideal body weight and oral Bu doses and Cy doses were calculated on an adjusted body weight. All patients were treated as inpatients and received our usual supportive care including transfusion support, pneumocystis prophylaxis, antiviral prophylaxis, growth factor support and broad-spectrum antibiotic coverage for febrile neutropenia. 26 consecutive patients > 60 with relapsed NHL underwent autologous SCT at Massachusetts General Hospital between 1995 and February 2005. The median age of the population was 66 (range 60–78) years. 17 (65%) of the 26 patients were females. 15 patients had large cell lymphoma, 2 immunoblastic lymphoma, 3 follicular lymphoma, and 6 mixed small cleaved cell and large cell. All patients had recurrent disease; 7 patients were in a CR and 19 patients in a PR at the time of transplant. The median time from diagnosis to transplant was 32 months. All patients engrafted. The median time to absolute neutrophil count > 500/uL was 10 days (range 9–11) days. The median time to platelet count > 50,000/uL unsupported was 15 (range 12–39) days. The 100-day transplant related mortality was 0%. Patients experienced the following serious toxicities: interstitial lung disease (n=1), seizures in a patient with CNS lymphoma (n=1), venoocclusive disease which resolved (n=2), and transient atrial fibrillation (n=3). With a median follow-up of 22 months (range 4 months–9.5 years), the two year overall survival is 63% and two year progression-free survival is 42%. Six patients above the age of 70 years have been transplanted. 100-day transplant related mortality was 0%. With a median follow up of 25 months, all 6 patients are alive and 3 of 6 are progression free. In conclusion, 1) Bu/Cy is a well-tolerated conditioning regimen for patients with NHL above the age of 60 years, including selected patients over the age of 70; 2) Age alone should therefore not be used as an exclusion criterion for autologous SCT.

Intermediate Dose Cyclophosphamide Followed by Sequential GM-CSF and G-CSF: An Efficient and Predictable PBPC Mobilization Regimen for Autografting: A Single Center Study of Two Hundred and Thirty Patients.

Collection of an optimal dose of peripheral blood progenitor cells (PBPC), eg, >5x106 CD34+ cells/kg, speeds engraftment after autologous bone marrow transplantation (ABMT). PBPC mobilization using high dose cyclophosphamide (Cy), eg 3–7gm/m2 and G-CSF typically produces a higher yield of PBPC than Cy or G-CSF alone, but WBC rebound following such regimens is often unpredictable, necessitating multiple assessments of blood WBC and CD34+ cell count, may require weekend leukapheresis (LP), and is associated with a high risk of febrile neutropenia. To minimize these problems while producing an adequate PBPC yield, we mobilized 230 unselected patients (pts) for ABMT using moderate dose Cy (1.5g/m2, day 1), followed by sequential administration of GM-CSF (500mcg/d, days 3–7) and G-CSF (5mcg/kg/d, day 8 until completion of LPs). This “CyGMG” regimen was based upon reports suggesting a synergy between GM-CSF and G-CSF. LP was initiated on day 11 irrespective of WBC or blood CD34+ cell count. Cy was administered on Friday (day 1) with LP starting on Monday (day 11 = LP day 1) and 20L LPs were performed for up to six days, thus avoiding weekend LP in most pts (median #LP = 3, range 1–6). Pt median age was 53 (range 19–78); 134 male, 96 female; diagnosis; myeloma (77), NHL (94), breast cancer (17), Hodgkin's disease (28), Testicular cancer (4), other (10). Median prior chemotherapy (CT) regimens = 2 (range 0–6). The estimated (Kaplan-Meier) cumulative probability of achieving a target collection of >2, or 5x106/CD34+ cells/kg on LP days 1–5 was 0.5, 0.77, 0.87, 0.91, 0.93, 0.87 and 0.25, 0.5, 0.65, 0.72, and 0.74 respectively. In addition, since 12/2003 when the collection target for pts with myeloma was increased to 10x106 CD34+ cells/kg, 76% of myeloma pts achieved this goal. Based on multivariate cox regression, diagnosis (myeloma vs other) and day 1 platelet (plt) count were significantly associated with achieving 2 or 5 x 106/CD34+ cells/kg and the above factors plus the # of prior CT regimens were associated with achieving 10x106/CD34+ cells/kg. However, (in contrast with a previous report) the day 1 plt count was not correlated with CD34+ cells/kg in the subgroup of myeloma pts (r=0.07, p=0.62). For non-myeloma pts a plt count >75,000 predicted achievement of 5x106 CD34+ cells/kg (2/17 pts with <75K plt vs 91/136 pts with >75K plt; p<.0001 by X2). Toxicities consisted mostly of mild bone pain and fevers, and 12 patients required hospital care during mobilization (not necessarily regimen related).Conclusion: This large series indicates that the above mobilization regimen (1) efficiently mobilizes adequate PBPC in the vast majority of an unselected population of pts for ABMT (including myeloma pts with a target dose of 10x106 CD34/kg), (2) obviates the need for WBC and peripheral blood CD34+ cell count monitoring before commencing LP and the need for weekend LP, and (3) is well tolerated by pts.

Clinical Tumor Responses Despite Graft Rejection after Nonmyeloablative Conditioning and Transplantation of Partially HLA-Mismatched (Haploidentical) Bone Marrow.

A variety of hematologic malignancies that are resistant to standard chemotherapy can be treated by nonmyelaoblative allogeneic stem cell transplantation, or NST. At our institution, 84 patients have enrolled onto sequential Phase I and II protocols of NST for hematologic malignancies utilizing haploidentical donors. Patients received non-T cell-depleted marrow after conditioning with fludarabine 30 mg/m2 (days −6 to −2), cyclophosphamide (Cy; 14.5 mg/kg/d, days −6 and −5) and 200 cGy total body irradiation on day −1. Novel post-transplantation immunosuppression included high dose Cy (50 mg/kg/d on day 3 only or (n=23) days 3 and 4 (n=40), mycophenolate mofetil from days 5–35 and FK-506 from day 5–180. Sixteen patients (19%) experienced graft rejection, defined as absence of donor chimerism on day 60, including 6/10 patients with myelodysplastic syndrome (MDS), 3/7 patients with chronic myeloid leukemia (CML), 2/3 patients with chronic myelomonocytic leukemia (CMML), 2/21 patients with acute myeloid leukemia (AML), and three patients with lymphoid malignancies. Fourteen patients recovered autologous hematopoiesis, whereas two (1 MDS, 1 NHL) died in aplasia. Interestingly, objective clinical responses were observed in 3/6 of the rejectors with MDS (1 CR, 2 PR), 2/3 rejectors with CML (2 molecular CR), and in both rejectors with CMML (1 CR, 1 PR). Two of the five patients with MDS and blast counts of greater than 20% survived for greater than 2 years with a decrease in blast counts and need for transfusions. Both patients with CML had disease refractory to imatinib mesylate, and both achieved molecular complete responses following transplantation. One of the patients has relapsed, and the other remains in hematologic and cytogenetic complete remission, though in minimal molecular relapse, at 6+ months after transplantation. Of the patients with CMML, one achieved a morphologic and hematologic complete remission at six months after transplantation, and the other achieved improved blood counts and disappearance of marrow blasts before relapsing at six months after transplant. In summary, fatal graft rejection is uncommon following nonmyeloablative BMT with post-transplantation cyclophosphamide. Clinical responses occurred despite graft rejection, particularly among patients with chronic myeloid malignancies. Potential mechanisms of the anti-tumor effect include an effect of conditioning, a transient graft-versus-host reaction by donor T or NK cells, or an abrogation of host tolerance to tumor by either the graft-versus-host or host-versus-graft reaction.