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Fludarabine

Fludarabine phosphate

Thymoglobulin

Antilymphocyte Serum

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Immunosuppressive Agents

Immunologic Factors

Conditioning regimen consisting of fludarabine 30 mg/m2 on days -4, -3 and -2 (total dose 90 mg/m2), followed by a singe dose of 2 Gy TBI administered on day 0, at a low dose-rate (≈ 7 cGy/min), before infusion of cells.

Conditioning consisting of 8 Gy TLI and ATG. TLI will be administered by linear accelerator at a dose of 80 cGy daily, starting 11 days before transplantation, until a total of 10 doses (800 cGy) has been delivered. The irradiation will consist of a supradiaphragmatic mantle field, a subdiaphragmatic field including an inverted Y and splenic ports, encompassing all major lymphoid organs, including the thymus, spleen, and lymph nodes, as used in the treatment of Hodgkin's disease (Kaplan HS, Cancer Research 26:1268-1276, 1966). The Waldeyer ring is not included. ATG (Thymoglobulin®, Genzyme), at a dose of 1.5 mg/kg/d, will be given intravenously on days -11 through -7.

Conditioning regimen: TBI + Fludarabine

Conditioning regimen:TLI (8 Gy) + ATG

Frederic Baron, MD, PhD

Yves Beguin, MD, PhD

Johan Maertens, MD

Koen Theunissen, MD

Harry Schouten, MD

The present project aims at comparing two nonmyeloablative regimens currently used in 2 major HCT centers in the US for patients with HLA-matched related or unrelated donor: the one from the Seattle group consisting of 2 Gy TBI with fludarabine (90 mg/m²) versus the one from the Stanford group combining 8 Gy TLI with ATG.

I. OBJECTIVES

The present project aims at comparing two nonmyeloablative regimens currently used in 2 major HCT centers in the US for patients with HLA-matched related or unrelated donor: the one from the Seattle group consisting of 2 Gy TBI with fludarabine (90 mg/m²) versus the one from the Stanford group combining 8 Gy TLI with ATG.

II. DESIGN OF THE STUDY

The study is a multicenter, randomized phase II study, comparing two conditioning regimens. Sixty patients with HLA-matched donors will be randomized between the TBI or the DLI regimen. There will be a stratification between centers. There will be a stopping rule for graft rejection \> 15% at day 180 (in each group separately), and for nonrelapse mortality \> 35% at day 180 (in each group separately). If the stopping rules are not triggered after 60 patients and no statistically significant differences are seen between the 2 arms in terms of acute GVHD, graft rejection and survival, a second cohort of 40 patients will be included.

III. TREATMENT PLAN

III.1. Pre-transplant procedures Peripheral blood mononucleated cells from the patient as well as from the donor will be collected before conditioning, as per standard practice for all routine allogeneic HSC transplants. Part will be cryopreserved in 10 % DMSO and stored at -180°C in liquid nitrogen. The other part will be devoted to identification of specific donor and patient markers to be used in later measurements of chimerism.

III.2. Conditioning regimens The conditioning regimens used will be either the one developed in Seattle (TBI arm) or the one developed by the Stanford group (TLI arm). These 2 regimens have been extensively reported in major medical journals. In the TBI arm, conditioning will consist of fludarabine 30 mg/m2 on days -4, -3 and -2 (total dose 90 mg/m2), followed by a singe dose of 2 Gy TBI administered on day 0, at a low dose-rate (≈ 7 cGy/min), before infusion of cells. In the TLI arm, conditioning will consist of 8 Gy TLI and ATG. TLI will be administered by linear accelerator at a dose of 80 cGy daily, starting 11 days before transplantation, until a total of 10 doses (800 cGy) has been delivered. The irradiation will consist of a supradiaphragmatic mantle field, a subdiaphragmatic field including an inverted Y and splenic ports, encompassing all major lymphoid organs, including the thymus, spleen, and lymph nodes, as used in the treatment of Hodgkin's disease (Kaplan HS, Cancer Research 26:1268-1276, 1966). The Waldeyer ring is not included. ATG (Thymoglobulin®, Genzyme), at a dose of 1.5 mg/kg/d, will be given intravenously on days -11 through -7.

III.3. PBSC collection and transplantation PBSC mobilization and collection in the donor will be performed as per standard practice for all routine allogeneic HSC transplants. This is briefly described below.

The donor will be given SC injections of G-CSF at a dose of 10-15 µg/kg for 6 days (days -5 through 0). Additional doses of G-CSF may be given on days +1 and +2 if the first 2 leukaphereses do not yield sufficient numbers of CD34+ cells. G-CSF will generally be administered :

* In the evening on days -5, -4, -3, -2;
* Before 7:00 on days -1 and 0 (and on days 1 and 2 if necessary). Collection of PBSC will be carried out on day -1 and in the morning on day 0. Leukaphereses will be performed using a continuous flow blood cell separator and following a mononuclear cell collection protocol. The volume of blood processed will be 15-20 liters if donor is an adult or 10 liters/m2 if donor is a child. Anticoagulation will be performed with the ACD-A solution. The PBSC from the first day of harvest will be stored overnight at 4°C in the patient's own plasma. After the second harvest, PBSC from the first and second day of harvest will be infused into the patient.

Based on previous reports suggesting that higher dose of CD34 cells are associated with better outcomes after nonmyeloablative HCT, high doses of CD34+ cells (\>6.5 x 106/kg) should be ideally administered. Nevertheless, to limit donor procedures, only two leukaphereses are required. However, in case the required minimal number of cells (3 x 106 CD34+ cells/kg recipient) cannot be obtained with the first two collections, additional leukaphereses should be carried out unless contra-indicated for the donor.

Cells will be infused through a central catheter according to standard procedures.

III.4. Other treatments of the recipient

III.4.1. Immunosuppressive therapy The immunosuppressive regimens used will be the one used in standard practice for routine NM-HCT at our centers, i.e. an association of tacrolimus and mycophenolate mofetil (MMF).

MMF will be administered orally from the evening of day 0 through day 28 (sibling recipients) or day 42 (alternative donor recipients) at the dose of 15 mg/kg t.i.d.

Tacrolimus will be given orally at the dose of 0.06 mg/kg bid starting on day -3. The dose will then be adapted according to through whole blood values following standard procedures (between 15 and 20 ng/ml the first 28 days and between 10-15 ng/ml thereafter). Full doses will be given until day 100 (sibling recipients) or 180 (alternative donor recipients). Doses will then be progressively tapered to be definitely discontinued by day 180 (sibling donors) or 365 (alternative donor recipients) in the absence of GVHD. Tacrolimus may be stopped earlier in case of disease progression or graft rejection or continued longer in case of low donor T-cell chimerism or GVHD.

GVHD will be assessed according to standard criteria. Therapy for acute or chronic GVHD will use standard procedures/current protocols.

III.4.2. Growth factors Growth factors will be used as per standard practice for all routine NM-HCT. No myeloid growth factor will be administered unless the granulocyte count falls below 1000/µl. Patients may then be treated with 5 µg/kg/day of G-CSF to maintain the granulocyte count \> 1,000/µl. Erythropoietin may be administered as required.

III.4.3. Infection prophylaxis Infection prophylaxis against bacterial, fungal, viral and parasitic agents will be carried out as per standard practice for all routine NM-HCT.

III.4.4. Donor lymphocyte infusion Donor lymphocyte infusion (DLI) will be given as per standard practice for all routine NM-HCT. DLI may be given in case of poor T-cell chimerism or disease progression according to standard procedures/current protocols.

IV. PATIENTS' FOLLOW-UP

IV.1. Quality controls of cell products

IV.1.1. Peripheral blood of donor on days of PBSC collection As per standard practice for all routine allogeneic PBSC transplants.

IV.1.2. Leukapheresis product As per standard practice for all routine allogeneic PBSC transplants including determination of the number of TNC, CD34, CD3 , CD4 and CD8 cells transplanted.

IV.2. Toxicities of cell infusions Potential toxicities associated with PBSC infusions will be carefully monitored per the standard procedures.

IV.3. Chimerism The chimeric status of hematopoietic cells will be carefully monitored post-transplant, as per standard practice for all routine allogeneic transplants. Donor chimerism will be measured in whole blood as well as bone marrow. In addition, peripheral blood cells will be separated by RosetteSep procedure (Stem Cell Technologies, Vancouver, Canada) to determine the proportion of donor and recipient cells in pure population of T (CD3+) cells. Fluorescent in-situ hybridization (FISH) for X and Y chromosome will be used preferentially for sex-mismatched HCT, while PCR techniques based on short tandem repeat (STR) markers will be used for sex-matched HCT. Pre-transplant donor and recipient peripheral WBC will serve to identify specific markers.

We define complete chimerism as the presence of \>95% of T cells of donor origin and mixed chimerism as the presence of 6-94% of T cells of donor origin. Graft rejection is defined as the occurrence of T cell chimerism \< 5% and engraftment as the occurrence of more than 5% T cells of donor origin in the first month following the transplant.

The proportion of donor chimerism will be determined at the following time points:

1. peripheral blood :

   * Days 28, 42, 60, 100, 180 and 365 post-transplant : whole blood and CD3+ cells;
   * Analyses on day 60 are only necessary when chimerism is \< 80% on days 28 and/or 42;
   * Analyses on whole blood on days 42, 100, 180 and 365 are only necessary when bone marrow analyses are not feasible/successful.
2. bone marrow : • Days 42, 100, 180 and 365 post-transplant : whole bone marrow.

IV.4. Clinical data Patient will be carefully observed and the following clinical parameters will be recorded (see appendices B and C). Appendices B and C should be send not more than 3 months after the patient achieved the target day after HSCT (day 100, day 180, 1-yr, 2-yr, 3-yr, 4-yr and 5-yr) to Frederic Baron at the fax # 32-4-366 8855.

* Incidence, timing and severity of acute GVHD, its treatment and outcome;
* Incidence, timing and severity of chronic GVHD, its treatment and outcome;
* Incidence, timing and severity of cytopenia, its treatment and outcome; number of platelet and RBC transfusions; G-CSF usage;
* Time to achieve 500 neutrophils, 1000 neutrophils, 20 000 platelets and 50 000 platelets;
* Duration of hospitalization, if any;
* Incidence of bacterial infections;
* Incidence of fungal infections;
* Incidence of CMV infections (by quantitative PCR) and CMV disease;
* Incidence of other viral infections;
* Incidence of other infections;
* Evolution of the primary malignant disease : response, relapse, its treatment and outcome;
* Any other serious complication associated with the transplant procedure;
* Death and survival.

IV.5. Immunologic data. In patients transplanted at the university of Liège, immune reconstitution in the patient will be monitored as per standard practice at ULg for all routine allogeneic transplants. For patients transplanted outside of the University of Liège and willing to participate to the immune recovery study, 50 mL of fresh heparinized blood collected on days 42, 100, 180, 365 and 730 can be send at room temperature to Olivier Dengis, Department of Clinical Hematology, CHU Sart-Tilman, B4000 Liège.

Allogeneic HCT With HLA-matched Donors : a Phase II Randomized Study Comparing 2 Nonmyeloablative Conditionings

Allogeneic Hematopoietic Cell Transplantation With HLA-matched Donors : a Phase II Randomized Study Comparing 2 Nonmyeloablative Conditionings

Percentage of participants with aGVHD according grades:

Grade I: rash skin \< 25 % area; bilirubin: 20-30 mg/ml; diarrhea: 500-1000 ml/day Grade II: rash skin 25-50 % area; bilirubin: 30-60 mg/ml; diarrhea: 10000-1500 ml/day Grade III:rash skin \> 50 % area; bilirubin: 60-150 mg/ml; diarrhea: \>1500 ml/day Grade IV: erythroderma; bilirubin: \> 150 mg/ml; diarrhea: \>2000 ml/day

Grade IV is the worst grade Patients given a second allogeneic HCT were censured for GVHD analyses.

graft rejection are reported in outcome measure data table (defined as ≤ 5% donor chimerism in T cells, total white blood cells and/or total bone marrow cells).

Comparaison of the number of Participants with chronic GVHD in the 2 groups

Analyses of ATG levels in order to assess the immune system recuperation

Maastricht University Medical Center

KU Leuven

University of Liege

Prof

Conditioning regimen consisting of fludarabine 30 mg/m2 on days -4, -3 and -2 (total dose 90 mg/m2), followed by a singe dose of 2 Gy TBI administered on day 0, at a low dose-rate (≈ 7 cGy/min), before infusion of cells.

Conditioning regimen TBI + Fludarabine: 2 Gy TBI, Fludarabine 90 mg/m²

Flu-TBI

Conditioning consisting of 8 Gy TLI and ATG. TLI will be administered by linear accelerator at a dose of 80 cGy daily, starting 11 days before transplantation, until a total of 10 doses (800 cGy) has been delivered. The irradiation will consist of a supradiaphragmatic mantle field, a subdiaphragmatic field including an inverted Y and splenic ports, encompassing all major lymphoid organs, including the thymus, spleen, and lymph nodes, as used in the treatment of Hodgkin's disease (Kaplan HS, Cancer Research 26:1268-1276, 1966). The Waldeyer ring is not included. ATG (Thymoglobulin®, Genzyme), at a dose of 1.5 mg/kg/d, will be given intravenously on days -11 through -7.

Conditioning regimen II (TLI 8 Gy + ATG \[Thymoglobulin\]): TLI 8 Gy + ATG (Thymoglobulin) 7.5 mg/kg

TLI-ATG

Total

Age, Categorical

Age, Continuous

Sex: Female, Male

Netherlands

Belgium

Region of Enrollment

HLA-identical sibling

10/10 HLA-allele matched URD

Donor: HLA-identical sibling/ 10/10 HLA-allele matched URD

13/107 included patients (6 in the Flu-TBI and 7 in the TLI-ATG arm) were excluded from analysis because they did not meet the inclusion criteria at the time of the start of the conditioning (disease relapse (5), ineligible for further irradiation (3), donor refusal (2), HLA-mismatched donor (2), and poor PS (1)).

Dr. Frédéric Baron

Patients given a second allogeneic HCT were censured for GVHD analyses.

Percentage of Participants With Grade II-IV Acute GVHD Between the 2 Groups

Number of Participants With Graft Rejection as Defined by Whole Blood and T Cell Chimerism

Percentage of Participants With Chronic GVHD in the 2 Groups

19 of 49 Flu-TBI patients (39%) versus 25 of 45 TLI ATG patients (56%) had a least one episode of bacterial infection the first 100 days after transplantation (P = 0.15).

For fungal infections, the figures were 3 of 45 (6%) and 7 of 45 (16%), respectively (P = 0.19)

Among CMV-seropositive patients and/or donors, the 100-day cumulative incidence of CMV reactivation was 31% in Flu-TBI patients versus 47% in TLI-ATG patient

Bacterial infection

Fungal infection

CMV reactivation

Incidences of Bacterial, Fungal and Viral Infections in the 2 Groups.

Four-year cumulative incidences of relapse/progression were 22% and 50% in Flu-TBI and TLI-ATG patients, respectively

Spots Global Cancer Trial Database for conditioning regimen:tli (8 gy) + atg