Allogenic Versus Autologous Stem Cell Transplantation in Adult Acute Myeloid Leukemia Patients with Normal Cytogenetics and FLT3 Negative State Thesis Submitted for Partial Fulfillment of MD Degree in Medical oncology BY Hossam El-deen Ahmed El-Ashtokhy

M.Sc. in Medical Oncology

Supervisors Dr. Hossam Mohamed Kamel Professor of Clinical Hematology National Cancer Institute, President of Cairo University

Dr. Mohamed Abdelmooti Mohamed

Dr. Yasser Hassan El Nahas

Professor of Medical Oncology

Professor of Clinical Pathology

National Cancer Institute,

National Cancer Institute,

Cairo University

Cairo University

Dr. Raafat Mohamed Abd El-Fattah Assistant Consultant of Medical Oncology National Cancer Institute, Cairo University National Cancer Institute, Cairo University

2015

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Abstract Introduction: Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal expansion of myeloid blasts in the peripheral blood (PB), bone marrow (BM) and/or other tissues. Stem cell transplantation (SCT) represents the only curative therapy for intermediate- and high-risk AML. Aim of the Work: To compare allogenic with autologous peripheral blood SCT (PBSCT) in adult normal karyotype (NK)-AML patients with FLT3 negative state regarding toxicities of transplant procedure, transplant-related mortality (TRM), diseases-free survival (DFS), and overall survival (OS). Patients and Methods: This study included patients with AML in CR1 who underwent PBSCT by CIRO National Cancer Institute (NCI) bone marrow transplantation (BMT) team at BMT units of Nasser Institute & El-Sheikh Zayed Hospitals with the following eligibility criteria: age 1650ys, PS < 2, with normal cytogenetics and FLT3 negative status in first complete remission (CR1). Results: Forty-three patients with the previously mentioned eligibility criteria were included & followed up in this study during the period from January 2011 to December 2014. For the whole group, the median age was 29 years. Thirty-four patients underwent allogeneic SCT while the 2

remaining 9 patients underwent autologous SCT. After a median follow up of 21.5 months (0.3– 46.5), the cumulative 2-year OS and DFS in the allogeneic group were 73.5% and 70.6% respectively, compared to 74.1% and 64.8% respectively in the autologous group with no statistically significant difference in OS and DFS between the two transplant groups (p = 0.690 and 0.768 respectively). Conclusion: The above-mentioned data in our study showed noninferiority of autologous compared to allogenic SCT in this category of patients. To confirm these results, larger studies are needed, with inclusion of a third arm (consolidation with chemotherapy), and new emerging molecular markers for better choice of the type of consolidation treatment. Key Words: Acute myeloid leukemia; Hematopoietic stem cell transplantation; Normal cytogenetics; FLT3.

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INTRODUCTION Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of myeloid blasts in the peripheral blood, bone marrow, and/or other tissues. It is the most common form of acute leukemia among adults and accounts for the largest number of annual deaths from leukemias in the United States, (Margaret et al, 2012). In Egypt, according to National Cancer Institute (NCI) registry, (Mokhtar et al, 2007), there were 1300 cases of leukemia attending the NCI, these cases accounted for 7% of all 18946 newly diagnosed proven malignant cases. In 2002, the number of patients with AML was found in 169 patients (1.8%), in 2003 the number of patients with AML was 180 patients (1.9%). Gharbiah population-based cancer registry in the years 2000-2002 reported that 487 cases of leukemia (acute and chronic) was registered with an average of 162 cases per year. The most frequent type of leukemia was AML with a frequency of 121 cases (over the years 2000-2002) representing 27.2% of all incident leukemias. (Ibrahim et al, 2007).

In 2008, WHO revised the diagnostic and response criteria for AML to include additional recurrent genetic abnormalities created by reciprocal translocations/inversions, and a new provisional category for some of the

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molecular markers that have been found to have prognostic impact, (Margaret et al, 2012).

Additionally, the category of AML with recurrent genetic abnormalities was expanded to include the following: t(9;1'l)(p22;q23), t(6;9)(p23;q34) (provisional entity), inv(3)(q21 q26.2) or inv(3;3)(q21;q26.2) (provisional entity), and t(1;22)(p13;q13) (provisional entity), in addition to the previously

recognized

t(8;21)(q22;q22);

inv(16)(p13;1q22)

or

t(16;16)(p13.1;q22); and t(15;17)(q22;q’l2) [APL subtype]. Other provisional entities include AML with molecular lesions such as mutated NPM1 or CEBPA genes, (Swerdlow et al, 2008). The

intermediate-risk

cytogenetic

category

is

the

most

heterogeneous group in AML, because it encompasses both normal karyotype without gross structural abnormalities and those with structural changes that are considered neither poor-risk nor favorable, (Grimwade et al, 1998; Byrd et al, 2002). However, even in patients with normal karyotype AML (NK-AML), clinical outcome is heterogeneous.

Molecular profiling is increasing the ability to identify mutations that carry prognostic impact. Thus, in addition to basic cytogenetic analysis, new molecular markers help to refine prognostics groups, particularly in patients with a normal karyotype. These markers include 5

FMS-like tyrosine kinase 3 (FLT3), c-KIT, nucleophosmin (NPM1), and CEBPA gene mutations (Bienz et al, 2005; Verhaak et al, 2005)

The 2 most frequent molecular lesions with prognostic impact in patients with AML are mutations of the NPM1 gene (28%-35%) (Thiede et al, 2006; Falini et al, 2005) , (Patel et al, 2012) encoding a shuttle protein within the nucleolus and mutations of the FLT3 gene (37%-46% of patients) encoding a receptor tyrosine kinase involved in hematopoiesis (Frohling et al, 2002; Schlenk et al, 2008; Patel et al, 2012). The median OS among patients with FLT3-ITD in the absence of a wild-type FLT3 was only 7 months compared with 46 months among both wild-type FLT3 patients with or without FLT3-ITD (Whitman et al, 2001). Allogeneic HSCT was beneficial for those with FLT3 internal tandem duplication (FLT3-ITD) or, in the absence of FLT3-ITD, for those without mutations in NPM1 and CEBPA, whereas for the subgroup with mutations in NPM1 and without FLT3-ITD there was no apparent benefit to having a matched sibling donor. However, testing for such novel genetic lesions, as well as whole genome analyses, RNA and microRNA profiles which have the potential to further refine AML risk are not yet in routine clinical use in many transplant centers. Also, these molecularly defined entities have not yet been robustly evaluated for outcome of allogeneic 6

HSCT, since the currently published molecular analyses have usually been done in highly selected subgroups of patients and in the context of retrospectively performed studies, (Mohty, 2012). The role of autologous HSCT in AML is still controversial. In general, the results of autograft for AML have remained constant over the last 10 years. There has been a reduction of NRM, probably due to better supportive therapy. In most studies, LFS ranges from 40 to 50% at 3 years. These results are encouraging, especially considering that autologous HSCT can be now offered to patients up to 65 years and beyond, (Breems; Lowenberg, 2007). Autologous HSCT is usually restricted to older AML patients, patients with APL in second molecular remission and younger patients lacking a sibling or unrelated donor. However, the picture may change in the future given the fact that the novel molecular and cytogenetic stratification methods may allow for identification of AML entities which could benefit from autografting as a post-remission therapy, (Mohty, 2012).

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PATIENTS AND METHODS This study included patients with AML who underwent PBSCT by NCI BMT team at BMT units of Nasser Institute and El-Sheikh Zayed Hospital with all the following patient's eligibility criteria: 1. Age: 16-50ys 2. Normal cytogenetics, FLT3 negative. 3. In first complete remission (CR1) according to revised recommendations of the international working group for diagnosis, standardization of response criteria, treatment outcomes, and reporting standards for therapeutic trials in AML (Cheson et al, 2003). 4. Having HLA-identical family donor (in case of allogenic SCT only). 5. ECOG performance status < 2. 6. Written informed consent.

Donor Eligibility (in case of allogenic SCT): 1. HLA identical family donor. 2. Good general condition. 3. Written informed consent.

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Pretransplant assessment: Detailed evaluation of the marrow transplant candidates as well as their donors was performed before proceeding to the transplant procedures. For all patients an informed consent has been obtained from them or their first degree relative after explaining all aspects of the technique of allogeneic and autologous stem cell transplantation.

The pretransplant recipient evaluation included:  Review of prior medical information including previous chemotherapy treatment.  Laboratory tests including: 1- HLA-typing. 2- Complete blood count including platelets and reticulocytes count. 3- Chemistry tests to assess the liver and kidney functions as well as blood glucose level and serum uric acid. 4- ABO and Rh typing. 5- Bone marrow aspirate and/or biopsy. 6- Diagnostic spinal tap was performed in patients at risk of CNS disease. 7- Serological screening tests for viral hepatitis, CMV, and HIV for evidence of recent infection or previous exposure to these 9

viruses, and Patients with evidence of recent infection were postponed till resolution of infection.  CT chest, abdomen & pelvis were performed in all patients.  ECG and echocardiography.  ENT and Dental examination for exclusion of septic foci.

The pretransplant donor evaluation (in case of allogenic SCT only): All donors were siblings of the patients. In addition to the serological testing for HLA typing, the donors were subjected to complete medical assessment including:  Complete blood count and chemistry tests to assess different organ function.  Bone marrow examination for assessment of cellularity.  ABO and Rh typing.  Serological tests for recent or previous viral exposure were the same as for recipients.

The transplantation procedures 1. Venous access Before isolation of the patients a central venous catheter was inserted by the vascular surgeon to facilitate the administration of PBSCs, blood products, fluids and other intravenous medications as well as total parenteral nutrition (TPN) solutions (if indicated). 10

Single lumen right atrial Hickman infusion catheter was usually inserted in allogenic patients and a double lumen Hickman apheresis catheter was inserted in autologous patients. The Hickman catheter was inserted via the internal jugular vein and fed out through a subcutaneous tunnel in the anterior chest wall. This extravascular part provides an excellent mechanical barrier against microbial contamination of the blood stream. 2. PBSC mobilization: Group I: (filgrastim-mobilized allogeneic PBSCs): - Donor PBSC mobilization was done using filgrastim (Neupogen) 10 µg/Kg S.C. daily from day -4 to day 0. - Leukapheresis: was performed on day 0. - PBSCs were immediately infused to the patients at the day of collection Group II: (filgrastim-mobilized autologous PBSCs): - Patient PBSC mobilization was done using cyclophosphamide (CPA) 1.5 gm/m2 IV infusion over 2 hours with Mesna (Day l= C1) and filgrastim (Neupogen) 5µgm/kg/12 hours S.C. daily from the sixth day of cyclophosphamide (C6) to the last day of leukapheresis (C10 ±C11 and C12) with daily blood counts. - Leukapheresis: was performed on C10 ± 11 ± 12 according to CD34+ cell count. - PBSCs were cryopreserved for 2-3 weeks till their re-infusion. 3- Leukapheresis After mobilization, the PBSC were collected from donors through peripheral veins (in allogeneic setting) or from patients through the apheresis catheter (in the autologous setting) using computerized continuous flow cell separator (COBE) During the collection procedure, the whole blood was pumped into the cell separator at inlet flow rates ranging between 50-80 ml/min, and mixed 11

with anticoagulant [acid citrate dextrose formula-A (ACD-A)] with an inlet to ACD-A ratio of 12:1. About 10-12 liters of blood were processed, resulting in the infusion of 800- 1000 ml of ACD – A during a period of 120-240 minutes. Donors (or patients) were given 1 gm of effervescent calcium dissolved in 200 ml water twice daily during the days of leukapheresis to prevent ACD-A induced hypocalcaemia. During collection procedure, vital signs were monitored closely at the beginning of the procedure and at least every 30 minutes during the collection. Donors (or patients in autologous group) were questioned and observed for symptoms of hypocalcaemia as perioral tingling which was managed by slowing the rate of blood flow, adding saline to return line and slow IV administration of calcium gluconate. As serial PBSC collections may be associated with platelet loss and decrease in hematocrit, close monitoring of blood counts between PBSC collections was performed. Also daily measurements of serum calcium level were done. All donors (and patients in autologous group) were discharged from the hospital the day following leukapheresis. No significant donor complications were reported.

4- Analysis ofCD34+ Cells Fresh PBSC harvest aliquots were immunostained and analyzed for CD34+ cells aiming to collect at least a total of 3 x 106 /kg BW CD34+ cells after a maximum of three leukapheresis in the autologous setting. In allogeneic transplant, we planned to restrict the amount of the CD34+ cells between 3-7 X 106/kg of the patients' BW to assure adequate engraftment and to decrease the incidence of GVHD by limiting amount of T-cells infused.

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Fresh peripheral stem cell harvest aliquots were immunostained and analyzed within 24 h. CD34 positive cell levels were determined flow cytometrically using two-color immunofluorescence labelling by whole blood method. Cells were analyzed using FAGS scan flow cytometer (BD Monoclonal Center Cells were labelled with fluorescein-conjugate anti-CD34. Conjugated non-immune isotype was run as negative control Ungated data was initially collected and then re-analyzed by a gated analysis on the "lymphoid" cells as defined by the side scatter peak of the lymphocyte population. Gated percentages of CD34+/CD33- and CD34+/CD33+ were converted to percentage of total ceils analyzed and then multiples by absolute mononuclear cell number to yield an absolute CD 34+/CD33- and CD34+/CD33+ result. 5- Cryopreservation (in autologous setting only) a) Each 50ml of the collected PBSC was transferred to a separate stem cell cryopreservation bag (Fenwell® Cryocyte bag, 500 ml volume) using the attached tubing. b) Using ice flecks, to keep PBSC product cool, to each Cryocyte bag an equal amount of cryopreservation mixture (table 18) was added. All air bubbles from the freezer bags were then carefully removed, and then the tubings were clamped (Rowley, 1992). c) Full identifying information including patient's full name, hospital number, PBSC collection date and volume and unique number for each freezing bag had to be written on the freezing bag label. d) The tubing of the bag were sealed twice, and the seal was cut at the second seal away from the bag. e) The freezing bag was then placed flat without bending edges or ports in an appropriately sized metal storage canister.

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Table (18): Cryopreservation mixture used in this study. Constituent

Material used

Concentration in Final cryopreservation concentration mixture

1-Cryoprotectant Dimethyl sulfoxide (DMSO) 2- Protein Autologous plasma

20%

10%

40%

20%

3- Culture media

40%

20%

Hank's balanced salt solution

(Rowley, 1992) f) Freezing of the product had to begin as soon as possible after addition of Cryopreservation mixture. Canisters containing the PBSC were placed in the freezing chamber of Planner 10® Series II controlledrate freezer. An automated program for this equipment, which results in a slow cooling rate of 1.5° C to 3.5° C per minute was used. g) After removal from the controlled-rate freezer, stem cell bags were stored in a monitored freezer in the liquid phase of liquid nitrogen at -196°C.

5.

Isolation and Peritransplant Medications Allogeneic patients started the isolation measures and conditioning

regimen during the week before the day of leukapheresis so that the last day of conditioning chemotherapy should precede the leukapheresis and stem cell infusion by at least 48 hours. In autologous sitting, the conditioning program started after full neutrophil and platelet recovery (usually 2-3 weeks from the start of mobilization). All patients were nursed under strict protective isolation in barrier nursing rooms with HEPA-filtered air. Attendants to the barrier nursing rooms wore gowns, head covers, masks and gloves. All materials introduced into the isolation unit were sterilized. 14

Nursing care during Isolation 

Daily weight and vital signs every 4 hours.



Central venous pressure measurements every 8 hours.



Daily fluid balance.



Hickman line care.

Results Forty-three

adult

cytogenetics, remission

patients

and

FLT3

(CR1), the

2014.

They

underwent

National

period

Cancer

status

included

during

AML,

negative

were

study

by

with

from

and

Institute

BMT

in

first

followed

January

peripheral

having

2011

stem

cell

team

at

normal complete

up

in

to

this

December

transplantation BMT

units

of

Nasser institute and El-Sheikh Zayed Hospital.

Patients the

availability

transplant stem

cell

only

9

of

groups:

genetically HLA

patients

groups

in

between was

the

patients

to

the

of

high

donor

in

the

group

and

autologous

numbers

according

sibling

(allo-SCT)

the

due

randomized

matching

Thirty-four

transplantation

discrepancy two

were

SCT patients

availability

to

into

2

allogeneic the

other

group.

The

between

the

of

matched

sibling donor in Egyptian patients due to their large family size.

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