A Phase II Trial of Non-Myeloablative Conditioning and

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A Phase II Trial of Non-Myeloablative Conditioning and

Transcript Of A Phase II Trial of Non-Myeloablative Conditioning and

A Phase II Trial of Non-Myeloablative Conditioning and Transplantation of Partially HLA-Mismatched and HLA-Matched Bone Marrow for Patients with
Sickle Cell Anemia and Other Hemoglobinopathies

Protocol Number: J0676

IRB Application#: NA_00002479

Principal Investigator:

Javier Bolaños Meade, MD Office: 410-614-6398 Pager: 410-283-8683 Email: [email protected]

Co-Principal Investigator: Sophie Lanzkron, MD Office: 410- 502-7770 Email: [email protected]

Co-Investigators:

Ephraim Fuchs, MD Leo Luznik, M.D. Richard J. Jones, M.D. Mark Levis, MD Carol Ann Huff, MD Stephanie Terezakis, MD Kenneth Cooke, MD William Matsui, M.D. David Loeb, M.D. Marianna Zahurak, MS

Lode Swinnen, M.D.
Richard Ambinder, M.D., Ph.D.
Allen Chen, MD, PhD Robert Brodsky, M.D. Ivan M. Borrello, M.D. Chtistopher Gamper, MD, PhD Gary Rosner, ScD

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TABLE OF CONTENTS
SCHEMA………………………………………………………. 3 1.0 OBJECTIVES…………………………………………………. 4 2.0 BACKGROUND AND RATIONALE……………………….. 4-7 3.0 DRUG INFORMATION……………………………………… 7-12 4.0 PATIENT SELECTION……………………………………… 12-15 5.0 TREATMENT PLAN………………………………………… 15-20 6.0 PATIENT MONITORING………………………………….. 20-24 7.0 TOXICITIES TO BE MONITORED……………………….. 24-26 8.0 STUDY PARAMETERS…………………………………….. 26-27 9.0 DATA MANAGEMENT…………………………………….. 27-29 10.0 STATISTICAL CONSIDERATIONS……………………… 29 11.0 RISKS AND BENEFITS…………………………………….. 30 12.0 INFORMED CONSENT…………………………………….. 30-31 13.0 ON-STUDY DATE…………………………………………… 31 14.0 OFF-STUDY DATE…………………………………………. 31
REFERENCES………………………………………………. 32-33 APPENDICES………………………………………………. 34-35

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TREATMENT SCHEMA

Days -9

Thymoglobulin 0.5 mg/kg IV with pre-meds

Start steroid taper



Days -8,-7

Thymoglobulin 2 mg/kg IV qd with pre-meds

Days -6, -5

 Fludarabine 30 mg/M2 iv qd

Cyclophosphamide (CTX) 14.5 mg/kg IV qd*



Days –4 -2

Fludarabine 30 mg/M2 iv qd



Day –1

TBI 400 cGy



Day 0

Infuse bone marrow and start penicillin



Days 3, 4

CTX 50 mg/kg iv q d

Mesna 40 mg/kg iv q d**

(First dose of CTX must be administered 48-72 hr after infusion of marrow)



Day 5

Begin sirolimus (section 6.6)** and

MMF 15 mg/kg po tid with maximum daily dose 3 gm/d

Day 30 Day 35 Day 60 Day 180 Day 365
1 yr, 2 yrs

 Assess Chimerism in peripheral blood
 Discontinue MMF
 Assess Chimerism in peripheral blood
 Evaluate disease Assess Chimerism in peripheral blood
 Discontinue sirolimus
Evaluate disease Assess Chimerism in peripheral blood
 Evaluate disease Assess Chimerism in peripheral blood

* Refer to Section 5.3 for complete dosing instructions. ** Or as per institutional standards.

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1.0 OBJECTIVES 1.1 Obtain estimates of transplant-related mortality (TRM) and progression-free survival in patients with severe hemoglobinopathies receiving nonmyeloablative conditioning and transplantation of partially human leukocyte antigen (HLA)-mismatched bone marrow (haploidentical) from relatives (“mini-haploBMT”) as well as HLA-matched donors. 1.2 Characterize donor hematopoietic chimerism in peripheral blood at days ~30, ~60, and ~180 after mini-haploBMT. 1.3 Characterize hematologic and non-hematologic toxicities of minihaploBMT.
2.0 BACKGROUND
Allogeneic blood or marrow transplantation (alloBMT) is a curative therapy for a variety of hematologic disorders, including sickle cell disease and other hemoglobinopathies such as thalassemia. Even when it is clear that alloBMT can give to these patients an improvement in their disease1, myeloablative transplants have important toxicities and mortalities associated. Substantial progress has been made recently in the development of reduced intensity conditioning regimens that facilitate the sustained engraftment of donor marrow with reduced toxicity. Most of these regimens incorporate highly immunosuppressive purine analogues, such as fludarabine, which allow the reduction or elimination of myeloablative agents such as busulfan or total body irradiation without endangering the sustained engraftment of HLA-identical allogeneic stem cells. Preliminary results of non-myeloablative allogeneic stem cell transplantation, or NST, suggest that the procedure can be performed in patients who are ineligible for myeloablative alloBMT, and that sustained remissions of several hematologic malignancies can be obtained.
Despite the encouraging results in hematologic malignancies, the results of nonmyeloablative alloBMT in patients with hemoglobinopathies are less encouraging. Recently, Jacobsohn et al reported on 13 patients with non-malignant conditions undergoing nonmyeloablative alloBMT2. Engraftment was poor in patients with hemoglobinopathies as only one patient engrafted out of 4 patients. These findings have been duplicated in other small studies3,4. Also, the lack of suitable donors continues to be a limit to access to transplantation. Therefore, developing novel strategies that address the issue of expanding donor pool and have different immune suppression are of paramount relevance for the therapy of sickle cell disease.
In the past five years, we have been developing non-myeloablative conditioning regimens for transplantation of marrow from partially HLA-mismatched, or haploidentical, bone marrow from first-degree relatives. The main goal of J9966 (RPN 99-11-05-01) was to titrate the dose of pre- and post-transplantation cyclophosphamide (CTX), a potent immunosuppressive drug, given in conjunction with pre-transplantation fludarabine and total body irradiation (TBI), to achieve a regimen that had an acceptably low risk of graft rejection and GVHD, the two major complications of haploidentical BMT. All patients received mycophenolate mofetil and tacrolimus, beginning on day 4 or 5 and terminating on days 35 and 50-180, respectively, to reduce the incidence and severity of GVHD. The first cohort of three patients received no pre-transplantation CTX

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and 50 mg/kg CTX IV on day 3, and two of the patients rejected their grafts. A second cohort of 20 patients received 14.5 mg/kg CTX IV on days –6 and –5 in addition to 50 mg/kg IV on day 3. Of 18 evaluable patients, 13 patients had donor engraftment on day 60, but accrual of patients to this dose level was stopped because 8/13 patients developed severe GVHD, an incidence convincingly in excess of the stopping criterion of 20%. To reduce the incidence of GVHD, a third cohort of patients received an additional dose of CTX 50 mg/kg IV on day 4, and MMF dosing was increased from bid to tid, based upon pharmacokinetic data suggesting the need for more frequent dosing. Of seventeen evaluable patients so far, two patients have had non-fatal graft rejection, and only one patient treated according to the protocol has had severe GVHD (an additional patient developed severe GVHD after withdrawal of immunosuppression to treat relapse). Two patients have died of causes other than relapse: one from GVHD, and the other from disseminated fungal infection. Of the sixteen patients who have been followed up to 100 days for relapse, eight have relapsed at a median of 64 days (range 24-~100) after transplantation, and six patients are alive and disease free at a median of 206 days (range, 100-429 days [as of Feb 8, 2004]) following BMT.
In order to better judge the safety and efficacy of our non-myeloablative BMT protocol, the tables below compare the results of J9966, dose level 3, to the results of the four largest published trials of HLA-identical sibling peripheral blood versus bone marrow transplantation for early stage leukemia.

Engraftment Data Author N (PB/BM)

Median age ANC 500/mm3†

Plt 20K†

Plt 50K†

Blaise5
Bensing er6
Schmitz
7

48/52* 81/91
163/166

37/36 42/42
39/37

15/21 16/21
12/15

13/21 13/19
15/20

Couban
8

109/118

45/44

19/23

16/22

J9966

17

31

16

24

*Numbers represent: recipients of peripheral blood/recipients of bone marrow

†Time from transplantation to designated count, sustained without transfusion

15/26 NA 20/26 NA
31

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Outcomes data

Author

aGVHD II-IV (%)

aGVHD III-IV (%)

cGVHD (%)

TRM (%)

Relapse (%)

Blaise

45/42*

17/28

55/30

23/21†

6/11

Bensinger

64/57

15/12

46/35

21/30†

14/25

Schmitz

52/39

28/16

66/50

24/24*

12/7

Couban

44/44

26/18

40/30

7/16†

15/20

J9966

47

13

NA

13

50

*Numbers represent: recipients of peripheral blood/recipients of bone marrow

†Transplant-related mortality (TRM) or relapse over entire study (median f/u ~ 2 years)

†100 day mortality

Compared to the patients receiving HLA-identical sibling bone marrow following myeloablative conditioning, patients on J9966 were younger, took longer to engraft platelets, and had a substantially higher rate of relapse, but were similar in the time to neutrophil recovery, the incidence of GVHD, and transplant-related mortality (TRM). The higher rate of relapse for patients on J9966 may be attributable to a benefit of myeloablative conditioning in reducing the risk of relapse, or that patients on J9966 had advanced, poor prognosis hematologic malignancies, which relapse more frequently than early leukemias after alloBMT.
Since the toxicities of non-myeloablative haploidentical BMT were not known when the trial was written, eligibility for J9966 was restricted to patients with advanced, poor-risk hematologic malignancies, such as chronic myeloid leukemia in 2nd chronic phase, advanced myelodysplasia, acute leukemia in 2nd remission, and lymphoma in relapse after autologous BMT. We have now expanded eligibility to ‘standard risk’ hematologic malignancies in trial J0457, which is still on going.
Between J9966 and J0457, 56 patients with hematologic malignancies received cyclophosphamide 50 mg/kg IV, either once (on day 3) or twice (on days 3 and 4) after non-myeloablative conditioning and haploidentical bone marrow transplant. Most of these patients had advanced disease or failed a previous autologous transplant. All were conditioned as outpatients with fludarabine, cyclophosphamide, and total body irradiation, transplanted with non-T cell-depleted marrow, and treated with tacrolimus and mycophenolate mofetil beginning the day after the last dose of cyclophosphamide. The most interesting finding was that compared to patients receiving a single dose of post-transplant cyclophosphamide, those receiving two doses had significantly less grade II-IV aGVHD (43% vs. 78%; p=.01) and grade III-IV aGVHD (20% vs. 53%; p=.006) by day 200 after transplant. Death from GVHD occurred in 5/13 assessable patients receiving one dose versus 2/28 assessable patients receiving 2 doses of cyclophosphamide.
Since the data to date suggest that our treatment regimen may be as safe as HLAidentical sibling BMT after myeloablative conditioning, non-myeloablative haploidentical BMT may be considered a reasonable treatment option for patients who have hemoglobinopathies. Also, the novel use of post-transplant cyclophosphamide on top of the nonmyeloablative conditioning, emerges as an interesting option of immunotherapy to prevent graft rejection. Moreover, as cancer relapse is not a concern in the setting of sickle cell disease, engraftment with NST should be curative.

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In clinical transplantation, antithymocyte globulin (ATG) has been used extensively in conventional myeloablative and non-myeloablative conditioning regimens to facilitate engraftment in patients with sickle cell disease14. This effect is largely mediated by in vivo T-cell depletion produced by the ATG, similar to the effect of monoclonal T-cell antibodies in the murine model.
Given that sirolimus (as opposed to calcineurin inhibitors) has not been commonly associated with the posterior reversible encephalopathy syndrome (PRES), we decided to change the immunosupression, switching sirolimus for tacrolimus. Also, given the very encouraging results obtained after 10 patients, and given that few patients may have a suitable HLA-matched donor, we have decided to expand the protocol to include these patients.
As of June 2011 we have transplanted 11 patients on study and of these, 6 have engrafted (54%) with no transplant related mortality, only 1 case of mild acute GvHD and no cases of chronic GvHD. While these results are actually very good considering the fact that all these patients have been haploidentical, there is a clear need for improvement. Experimental data using high dose cyclophosphamide has clearly shown that in order to increase engraftment efficiency there are 2 clear strategies to follow: increase the intensity of the conditioning (such as increasing the dose of TBI) or increase the cell dose of the graft9, 10. Increasing the intensity of the conditioning can translate into increasing toxicities, therefore, we are interested on increasing the cell count in the graft. As with standard bone marrow harvest there may be a limit on the number of cells that can be harvested, “priming the marrow” with filgrastim comes as an attractive option11-13.Studies using bone marrow priming with filgrastin show that it is possible to double the number of nucleated cells from the marrow, and interestingly, high cell doses have not been associated with increased risk of GvHD or severe toxicities to the donor12, 13.
Recently published data have shown that on patients at high risk of graft failure receiving non-myeloablative bone marrow transplants, the use of slightly higher doses of total body irradiation improve the probabilities of engraftment17,18. Given that we still facing graft failures in the order of 30% in our clinical trial, we decided to increase the dose of TBI to 400cGy after discussions within the protocol team and with Radiation Oncology19. The higher dose may be associated with a higher incidence of sterility in men, and the patient has performed sperm banking and will be informed about this increased risk. However, we do not think will increase graft-versus-host disease rates given that our graft-versushost disease rates using high-dose cyclophosphamide are similar between myeloablative and non-myeloablative transplants20,21. Moreover. Published data showed that TBI at 400cGy can be safely done in transplants using low intensity conditioning without increasing toxicities even on patients receiving mis-matched unrelated transplants22,23.
3.0 DRUG INFORMATION
3.1 Fludarabine
Fludarabine phosphate is commercially available.

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Fludarabine phosphate is purine antimetabolite.that, after administration, undergoes rapid conversion in plasma to the nucleoside 2-fluoro ara-A (F-araA). F-araA subsequently enters cells where it is phosphorylated to F-araATP and the monophosphate F-araAMP. Once activated, F-araATP inhibits DNA polymerase and ribonucleotide reductase. The monophosphate F-araAMP, once incorporated into DNA, is an effective DNA chain terminator.
Fludarabine monophosphate, 50 mg/vial, is reconstituted with 2 ml of sterile water, resulting in a 25mg/ml solution. The desired dose is further diluted to concentrations of 0.04-1 mg/ml in normal saline or 5% dextrose (50-100ml) for injection and will be administered by IV infusion over 30 minutes or longer.
Following IV administration, the drug is metabolized to 2-F-araA and widely distributed in tissues. 2-F-araA is excreted primarily in urine and has a terminal elimination half-life of 7 to 12 hours.
Clinical toxicities of fludarabine monophosphate include: myelosuppression, primarily lymphopenia and granulocytopenia, alopecia, rash, dermatitis, nausea, vomiting, anorexia, stomatitis, diarrhea, somnolence, fatigue, peripheral neuropathy, mental status changes, cortical blindness, hepatocellular toxicity with elevation in serum transaminases, and interstitial pneumonitis. These effects are reversible when the drug is discontinued.
Fludarabine will be administered by IV infusion over 30 minutes in a dose of 30 mg/m2/day on days -6 to -2.
Fludara® will be dispensed by the Oncology Pharmacy and is produced by Berlex Pharmaceuticals.
3.2 Cyclophosphamide (Cytoxan®)
Cyclophosphamide is commercially available.
Cyclophosphamide is an alkylating agent which prevents cell division primarily by cross-linking DNA strands. Cyclophosphamide is cell cycle non-specific.
Cyclophosphamide for injection is available in 2000 mg vials which are reconstituted with 100 ml sterile water for injection. The concentration of the reconstituted product is 20 mg/ml. The calculated dose will be diluted further in 250-500 ml of Dextrose 5% in water. Each dose will be infused over 1-2 hr (depending on the total volume).
Clinical toxicities of cyclophosphamide include alopecia, nausea and vomiting, headache and dizziness, hemorrhagic cystitis, cardiotoxicity, immunosuppression,

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myelosuppression, pulmonary fibrosis, increased hepatic enzymes and syndrome of inappropriate anti-diuretic hormone (SIADH).
Cyclophosphamide will be dispensed by the Oncology Pharmacy and is produced by Mead Johnson Pharmaceuticals.
3.3 Mesna (sodium-2-mercapto ethane sulphonate)
Mesna is a prophylactic agent used to prevent hemorrhagic cystitis induced by the oxasophosphorines (cyclophosphamide and ifosphamide). It has no intrinsic cytotoxicity and no antagonistic effects on chemotherapy. Mesna binds with acrolein, the urotoxic metabolite produced by the oxasophosphorines, to produce a non-toxic thioether and slows the rate of acrolein formation by combining with 4-hydroxy metabolites of oxasophosphorines.
Mesna is available in 200 mg, 400 mg and 1000 mg vials containing a 100 mg/ml solution. Each dose of mesna will be diluted further in 50 ml of normal saline to be infused over 15 minutes (or as per institutional standards). Mesna dose will be based on the cyclophosphamide dose being given. The total daily dose of mesna is equal to 80% of the total daily dose of cyclophosphamide.
At the doses used for uroprotection mesna is virtually non-toxic. However, adverse effects which may be attributable to mesna include nausea and vomiting, diarrhea, abdominal pain, altered taste, rash, urticaria, headache, joint or limb pain, hypotension and fatigue.
Mesna will be dispensed by the Oncology Pharmacy and is produced by Mead Johnson Pharmaceuticals.
3.4 Sirolimus (rapamycin, Rapamune®)
Sirolimus is an immunosuppressant that inhibits cytokine-stimulated T-cell activation and proliferation, and also inhibits antibody formation. The mean bioavailability of sirolimus after administration of the tablet is ~27% higher than the oral solution. Sirolimus oral tablets are not bioequivalent to the oral solution. Clinical equivalence has been demonstrated at the 2-mg dose level; however, it is not known if higher doses are clinically equivalent on a mg to mg basis. a) Sirolimus oral solution: Sirolimus oral solution (1 mg/mL) should be stored protected from light and refrigerated at 2°C to 8°C (36°F to 46°F). For dilution, the appropriate dose should be measured using an amber oral syringe, then added to a glass or plastic container that holds at least 60 mL. Before taking the dose, it should be diluted with water or orange juice then taken immediately; it should not be diluted with grapefruit juice. The syringe should be discarded after one use. Sirolimus oral solution provided in bottles may develop a slight haze when

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refrigerated, which does not affect product quality; allow the product to stand at room temperature and shake gently until the haze disappears.
b) Sirolimus tablets: Sirolimus tablets are available in 1 mg and 2 mg tablets that cannot be crushed or broken. Sirolimus tablets should be stored at 20° to 25° C (68°–77°F), protected from light.
The most common adverse reactions of sirolimus are: peripheral edema, hypertriglyceridemia, hypercholesterolemia, hypertension, increased creatinine, constipation, abdominal pain, nausea, diarrhea, headache, fever, urinary tract infection, anemia, thrombocytopenia, arthralgia, pain. Adverse reactions that have resulted in rates of sirolimus discontinuation >5% were increased creatinine, hypertriglyceridemia, and thrombotic thrombocytopenic purpura (TTP) / thrombotic microangiopathy (TMA). Sirolimus toxicities are summarized:

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PatientsDoseGvhdCyclophosphamideSirolimus