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4.2 management of engraftment syndrome

Engraftment syndrome may occur at the time of neutrophil recovery. Its clinical manifestations include noninfectious fever, rash, and vascular leak causing non-cardiogenic pulmonary edema, weight gain, and renal insufficiency [101]. Diagnostic criteria and a treatment schema for engraftment syndrome are included in Appendix B.  
4.3 TREATMENT OF MIXED CHIMERISM OR PERSISTENT/PROGRESSIVE DISEASE POSTTRANSPLANT
4.3.1 Donor Lymphocyte Infusion 4.3.1.1 Patients with persistent or progressive malignancy post-HSCT, or mixed chimerism that does not improve after tapering or discontinuing immune suppression may be eligible to receive donor lymphocytes (donor lymphocyte infusion, or "DLI") if they can be obtained from their respective donor.  
4.3.1.2 Additional lymphocytes will be collected by apheresis from the patient's original stem cell donor, either in steady state (i.e. no donor therapy) or after filgrastim administration.
4.3.1.3 DLI may be administered alone or after chemotherapy. DLI may be sequentially administered for mixed chimerism or disease progression Recipients with mixed chimerism or disease progression may receive DLI consisting of unmanipulated donor T cells. Alternatively, in cases where additional donor stem cells are desired, the donor product may be administered without lymphocyte purification.  
4.3.1.4 Recipients with mixed chimerism at day +28 will undergo repeat chimerism assessment at day +42 (+/- 3 days) and every two weeks thereafter as deemed necessary. If there is evidence of declining donor chimerism on consecutive determinations or if <50% donor chimerism on whole blood, lymphoid, or myeloid subsets at a single determination, recipients without GVHD will be eligible to receive DLI. DLI may be repeated every 4 weeks with dose escalation as indicated above, until donor chimerism > 95% is achieved or until GVHD develops. Cyclosporine, tacrolimus, and sirolimus will be maintained at planned specific doses through day +100 for recipients with mixed chimerism while DLI are administered. Recipients with mixed chimerism beyond day +100 may undergo withdrawal of respective immunosuppressive agents (i.e. cyclosporine, tacrolimus, and/or sirolimus) over 28 days, followed by DLI administration at escalating doses as outlined.  
4.3.1.5 Recipients with disease progression and without GVHD will be eligible to receive DLI. DLI may be repeated every 4 weeks with dose escalation as indicated above, until donor chimerism > 95% is achieved or until GVHD develops. For disease progression occurring after day +42 and before day +100, cyclosporine will be withdrawn over 28 days prior to administration of the first DLI.  
4.3.2 Other Post-Transplant Therapy - In addition to DLI, persistent or progressive disease may be treated with any approved therapy thought to be in the best interest of the patient, such as chemotherapy, cytokines, or monoclonal antibodies. Alternatively, such patients may be offered therapy on other NCI protocols.  
4.3.3 For patients with bcr-abl (+) CML, AML or ALL who were previously receiving tyrosine kinase inhibitors (e.g. imatinib, nilotinib and dasatinib) prior to transplant may resume this therapy at Day 28.  
As part of a prospective study, Carpenter and colleagues at the Fred Hutchinson Cancer research Center in Seattle administered imatinib from the time of engraftment until 365 days after allogeneic stem cell transplant in 22 patients, 15 with Ph+ acute lymphoblastic leukemia = 15; chronic myelogenous leukemia = 7). Before day 90, adults (n = 19) tolerated a median average daily imatinib dose of 400 mg/d (range, 200-500 mg/d), and children (n = 3) tolerated 265 mg/m2/d (range, 200-290 mg/m2/ d). The most common adverse events related to imatinib administration were grade 1-3 nausea, emesis, and serum transaminase elevations. The conclusion of this trial was that imatinib could be safely administered early after myeloablative allogeneic HCT at a dose intensity comparable to that used in primary therapy [124]. 4.4 TREATMENT OF GRAFT-VERSUS-HOST DISEASE 4.4.1 In recipients with suspected GVHD, standard clinical criteria and biopsy findings (when clinically indicated) will be used to establish the diagnosis. Acute GVHD will be assessed according to 1994 Consensus Conference on Acute GVHD Grading criteria [106]. See Appendix C for details concerning the grading and management of acute GVHD. Chronic GVHD will be assessed according to 2005 Chronic GVHD Consensus Project [61]. See Appendix Ffor details concerning the assessment of chronic GVHD.  
4.4.2 Recipients with clinical Stage 1 or 2 (Grade I) GVHD of the skin without any other organ involvement will be treated with a topical corticosteroid cream.  
4.4.3 In general, recipients with  Grade II acute GVHD will be treated with high-dose, systemic corticosteroids.  
4.4.4 Recipients who fail to respond satisfactorily to corticosteroids will be considered for second-line immunosuppressive therapy, e.g., tacrolimus, mycophenolic acid, monoclonal antibodies, or experimental GVHD protocols, if they are available.  
4.5 MENSES SUPPRESSION AND CONTRACEPTION
(For a full description of menses suppression and contraception please refer to the NIH BMT Consortium Supportive Care Guidelines at: http://intranet.cc.nih.gov/bmt/clinicalcare/guidelines.shtml) 4.5.1 Pre-menopausal women who have not undergone hysterectomy will be placed on a monophasic oral contraceptive to provide both menses suppression and contraception. This therapy will begin with the first cycle of induction therapy and continue until platelet recovery after transplantation. (>50,000/mm3 without transfusion). Start treatment prior to the onset of thrombocytopenia usually when withdrawal bleeding begins. Once thrombocytopenia occurs therapy is less likely to be effective. As general recommendations:
- Prescribe Lo-Ovral (300mcg norgestrol and 30 mcg ethinyl estradiol)
- 1 tablet daily. Instruct patient not to take placebo tablets.  
- If the patient is on another oral contraceptive, switch her to Lo-Ovral If Lo-Ovral is started when the patient is bleeding and bleeding persists for more than 2-3 days, increase the dose to 1 tablet twice daily. If bleeding persists for more than an additional 2-3 days (total 5 to 6 days after starting hormones), consult the NIH Gynecology Consult Service. 4.5.2 Female transplant recipients will be advised to use contraception for at least 1 year after transplantation and to have their male partners use condoms. Male transplant recipients will be advised to use contraception, preferably condoms, for 1 year after transplantation.  
4.6 BLOOD PRODUCT SUPPORT  
4.6.1 Recipients will receive packed red blood cells and platelets as needed to maintain Hb >
8.0 gm/dl, and platelets > 10,000/mm3(or higher, if clinically indicated). All blood products except for the stem cell product and DLI will be irradiated.  
4.6.2 Leukocyte filters will be utilized for all blood and platelet transfusions to decrease sensitization to transfused leukocytes and decrease the risk of CMV infection [107].  
4.7 NUTRITIONAL SUPPORT
When mucositis or GVHD prevents adequate PO intake, parenteral hyperalimentation will be instituted and discontinued under the direction of the dietary service. Oral intake will resume when clinically appropriate under the supervision of the dietary service of the Clinical Center. 4.8 ANTI-EMETICS Anti-emetic usage will follow Clinical Center Guidelines as well as recommendations from the Pharmacy service. 4.9 HEMATOPOIETIC GROWTH FACTOR SUPPORT
All patients will start filgrastim 10g/kg/day on day 0 starting after the stem cell infusion. The dose will be given subcutaneously (SC). Filgrastim will be continued until the absolute neutrophil count (ANC) is > 5000/l for at least one day and > 1000/l for three consecutive days. Filgrastim may be resumed if ANC drops below 1000/l.
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5 biospecimen collection

5.1 CORRELATIVE STUDIES FOR RESEARCH 5.1.1 Immunological Studies 5.1.1.1 T-Cell Receptor Vβ Repertoire Analysis by CDR3 Spectratyping ((Hakim lab).  
5.1.1.1.1 40 ml of blood will be drawn from the donor and placed into yellow top ACD tubes at a time before administration of G-CSF for stem cell mobilization. This sample permits calculation of spectratype complexity index [53].  
5.1.1.1.2 50 ml of blood will be drawn into yellow top ACD tubes at 1, 3, 6, and 12 months post-transplant from the recipient (Hakim Lab).  
5.1.1.1.3 These samples will be ficolled and magnetic cell sorting will be used to separate the CD4+ and CD8+ T cell subsets. RNA will be isolated and RTPCR followed by Vβ spectratype analysis will be performed.
5.1.2 Correlation between recipient T-cell subpopulations, serum IL-7 and IL-15 levels
(Gress/Hakim & Mackall laboratories):  
5.1.2.1 Baseline evaluation of donor lymphocytes: aliquot of donor apheresis product containing ~50 x 106 mononuclear cells. Performed in Gress/Hakim lab (10/CRC 3-3288; 301-402-3627).  
5.1.2.2 For all subjects, in vitro studies of immune characterization will focus on separation of distinct cell subsets by multi-parameter FACS analysis
Specifically, peripheral blood mononuclear cells (PBMC) will be analyzed by flow cytometry for expression of markers indicative of hematopoietic lineage, immune functional subsets, and activation state.  
Cell subsets may be analyzed for T cell receptor rearrangement circles (TREC).  
Cells may be activated in vitro with a number of different stimuli including specific antigens and mitogens which are known to activate distinct pathways of T lymphocyte function. Assays may include T cell proliferation, cytokine production and gene expression, cytotoxic T lymphocyte generation, and antigen presenting cell function.  
The specific assays to be used in the on-going data analyses are subject to be modified, deleted or replaced as technology and knowledge in the field evolve during the course of the study without constituting a change in research aims.  
These assays apply also to those samples received from outside institutions. If a significant departure from this "Immune Characterization" is contemplated based on accumulated data, then the protocol and consent will be amended accordingly to cover the new line of investigation and its potential risks to subjects.  
5.1.2.3 60 ml heparinized whole blood for FACS to Gress/Hakim lab (10/CRC 3-3288; 301-402-3627). Baseline before start of respective (EPOCH-F/R or FLAG) induction chemotherapy; days +7, +14, +28, +60, +100; 6 months, 9 months, 12 months, 18 months, 24 months, annually thereafter, and at the onset of either acute and/or chronic GVHD.  
5.1.2.4 20 ml sodium heparinized whole blood (two 10 ml green top tube) for FACS to Gress/Hakim lab (10/CRC 3-3288). At end of each cycle of the respective induction regimen; day 0 following administration of transplant preparative regimen.
5.1.2.5 Recipient IL-7 and IL-15 levels, for correlation with NK and T-cell subpopulations (above): 10 ml whole blood (1 10 ml red top tube), to be sent to Frederick for storage. IL-7 levels will be performed in Mackall laboratory
(10/CRC 1-3940; 301-402-0215); IL-15 levels will be performed in Gress/
Hakim lab (10/CRC 3-3288; 301-402-3627). Baseline before start of the respective induction chemotherapy; immediately before the initiation of the conditioning regimen; day 0 following administration of transplant conditioning regimen; days +7, +14, +28, +60, +100; 6 months, 9 months, 12 months, 18 months, 24 months, and annually thereafter. Note: IL-7 levels will not include day +60 or time points after day 100.  
5.1.2.6 Lymphocyte subpopulations, IL-7, and IL-15 levels may be obtained at other time points of interest (e.g., development of GVHD, attainment of complete remission, progression of disease, etc…).
5.1.3 Correlation of monocyte cytokine expression patterns with GVHD in recipients (Fowler Laboratory, 10/CRC 3-3224; 301-594-4536):
5.1.3.1 40 ml of blood will be drawn in heparinized tubes on day +14 post-SCT. 20 ml of blood will be drawn in heparinized tubes; days +28, +60,+100 Samples will be sent to the Hakim lab. These samples will be ficolled, cultured overnight in media containing the golgi inhibitor monensin, and evaluated for monocyte surface markers and intracellular cytokine markers for CD14 monocyte production of IL-1 alpha, TNF alpha, and IL-15.  
5.1.3.2 50 ml of blood will be drawn into green top heparinized tubes on day 7 posttransplant. Samples will be sent to the Hakim Lab. These samples will be ficolled, and evaluated either fresh (day +7 sample) or after CD3, CD28 stimulation (day 6 sample) for T cell cytokine secretion profile. This assay will be by cytokine capture flow cytometry, with surface markers of CD4 and CD8 evaluated. Cytokine capture staining of these populations will evaluate both T1
(IL-2, IFN-) and T2 (IL-4, IL-10) cytokine secretion.  
5.1.4 Tumor acquisition for generation of activated tumor derived lymphocytes (Optional).  
5.1.4.1 An important research objective is to determine whether the curative aspect of transplantation involves an immune component that is specifically directed towards tumor cells. Identification of tumor reactive T cells generated in the allogeneic context may help in the development of new therapies to improve anti-tumor potency and is on ongoing project in ETIB (Hardy*).  
5.1.4.2 To facilitate this evaluation of tumor reactive T cells, patients will be asked to undergo an optional excisional or core biopsy procedure to obtain tumor tissue (i.e. lymph nodes or masses) or additional bone marrow prior to chemotherapy administration and potentially after transplant, if there is residual tumor.  
5.1.4.3 The excisional/core biopsy procedure will be primarily applicable to patients with solid tumor masses (lymphomas, plasmacytomas, etc.). The Surgical Consult Service and/or Interventional Radiology Department of NIH Clinical Center will evaluate each case for their capacity to obtain tumor tissue; only cases that they deem at low risk for complication and can be performed without general anesthesia will be asked to undergo the biopsy procedure.  
5.1.4.4 For all patients, an additional 5 ml (heparinized syringe) bone marrow aspirate will be obtained at the times scheduled for bone marrow examination as per protocol.
5.1.4.5 Biopsy or bone marrow specimen samples obtained from the O.R. or Interventional Radiology, or after the bone marrow aspiration procedure, respectively, will be sent to the Hakim Lab. This material will be utilized in subsequent in vitro T cell assays.  
5.1.4.6 Patients may be asked to undergo a one-to-two liter single line apheresis procedure for lymphocyte acquisition for comparison with the tumor-derived product.
5.1.5 Analysis of post-transplant antibodies that selectively recognize tumor cell surface antigens (Hakim Laboratory).  
5.1.5.1 Post-transplant antibodies that selectively recognize tumor cell surface antigens may be capable of mediating antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) and, thereby, contribute to the allogeneic GVT effect. In addition, these antibodies may serve as tools for the discovery of new targets for human monoclonal antibody therapy. Based on 03-C-0077, the Rader laboratory developed a sensitive flow cytometry assay for detection of post-transplant serum antibodies against B-CLL tumor cell surface antigens. In order to identify these antibodies and subsequently the tumor cell surface antigens they recognize, the Rader laboratory has generated human antibody libraries from post-transplant peripheral blood mononuclear cells (PBMCs) for selection on primary B-CLL tumor cells by phage display. The inclusion of post-transplant serum antibodies and PBMCs from B-CLL patients with (i) HLA-matched unrelated donors and (ii) different GVHD
prophylaxis regimens provides an important extension of this ongoing study.  
5.1.5.2 Only for accrued B-CLL patients on either GVHD prophylaxis regimen arm, 50 ml heparinized whole blood before start of induction chemotherapy as well as 20 ml heparinized whole blood on days 0, +60, +100; 6 months, 9 months, 12 months, 18 months, 24 months, and annually thereafter to be sent to the Rader laboratory (10/CRC 3-3248) to the attention of Dr. Baskar (301-451-8361).  
5.1.6 Analysis of changes in immune cell populations and molecular events in the target tissues of chronic GVHD (Gress Lab 10/CRC 3-3288 301-402-3627; 12C121 301-594-5340).  
5.1.6.1 The pathogenesis of chronic GVHD is poorly understood and no studies rigorously evaluated cellular and molecular changes that occur in the target tissues of chronic GVHD. Heterogeneity in patient and treatment related factors complicate the group comparisons. Prospectively designed study with planned sequential sample collection is the ideal format that would give the best chance of success in study of this complex problem.
We will focus on the skin and oral cavity as the two most common target organs of chronic GVHD. Standard 6 mm punch biopsies will be performed in the skin and buccal mucosa in all patients at the time of initiation of immunosuppressive taper prior to onset of clinical chronic GVHD. Another biopsy will be performed at the onset of clinical chronic GVHD and at 6 months posttransplant in patients who do not develop chronic GVHD by this time point.  
Whole saliva will be collected at the same time as the buccal mucosal biopsies are performed to evaluate the changes in the salivary proteome at the onset of chronic GVHD. The samples will be analyzed using a variety of methods including immunofluorescence and confocal microscopy, gene expression profiles, and protein based assays in order to better understand the reconstitution of resident immune cell populations following HSCT and how they change with the onset of chronic GVHD.  
5.1.7 Sample Management and Storage (For more information see section **11.7** for Experimental Transplantation and Immunology Branch Preclinical Service Policy for Sample Handling)  
5.1.7.1 All samples will be coded and the key will be available to a restricted number of investigators. However, the nature of the study requires that clinical correlation be feasible as part of hypothesis generation. No change in research subject risk is foreseen from the knowledge acquired from study data. However, if in the judgment of the PI, this should change in the course of the study, NCI IRB will be informed to evaluate the eventual need for modification in subject consent process or for re-contacting subjects.  
5.1.7.2 Coded samples will be stored frozen at -20 to -180 C in a single location under the restricted control of the Clinical Core laboratory of ETIB.
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6 data collection and evaluation

Data will be prospectively collected and entered in real time into the Cancer Center Clinical Data System database (NCI C3D database) (information at http://ccrtrials.nci.nih.gov). It is expected that clinical data be entered into C3D no later than after 10 business days of the occurrence. The NCI PI and research nurse will have access to these data via web access. Adverse Events will not be collected or reported to the IRB during the Standard Therapy (EPOCH or FLAG) administered prior to the preparative chemotherapy (conditioning) and transplant phase (if applicable) except grade 5 events. 6.1.1 Adverse Events during the Pre-Transplant conditioning therapy phase (Flu-Cy):  
6.1.1.1 Grade 1, 2, and 3 adverse events will not be recorded, with the exception of all Grade 3 events that are unexpected, but are possibly, probably or definitely related to the research 6.1.1.2 All expected and unexpected Grade 4 adverse events possibly, probably or definitely related to the research will be recorded with the exception of:
a) Blood/Marrow abnormalities (will include grade 3 & 4 hemolysis)
6.1.1.3 All grade 5 adverse events will be recorded regardless of attribution.  
6.1.2 Adverse Events during the Transplant phase (Day 0 to +180):  
6.1.2.1 Grade 1, 2 and 3 adverse events will not be recorded, with the exception of all Grade 3 events that are unexpected, but are possibly, probably or definitely related to the research.  
6.1.2.2 All expected and unexpected Grade 4 adverse events possibly, probably or definitely related to the research will be recorded with the exception of:
a) Blood/Marrow abnormalities (will include grade 3 &4 hemolysis and cytopenia related to graft failure)
6.1.2.3 All grade 5 adverse events will be recorded regardless of attribution.
6.1.3 Due to time distance from transplant conditioning and inconsistent patient monitoring once they leave NIH, adverse events grade 1-4 during the follow-up phase (beyond Day +180) will not be recorded.  
6.1.3.1 However, all patients will be monitored for GVHD and the dates of onset will be recorded for GVHD, which is the main and only relevant transplant related complication after day +180 6.1.3.2 Blood/Marrow abnormalities due to grade 3 &4 hemolysis and cytopenia related to graft failure will be recorded 6.1.3.3 All grade 5 adverse events will be recorded regardless of attribution.
6.1.4 Adverse Events once patient experiences progressive malignancy In the event of progressive malignancy only Grade 5 adverse events will be recorded. GVHD will also be recorded. 6.1.5 Data collection procedures 6.1.5.1 After obtaining Informed Consent, a file will be created in the database with standardized forms. The NCI C3D will maintain data for described endpoints in the protocol. The medical record will maintain complete records on each patient including any pertinent supplementary information obtained from outside laboratories, outside hospitals, radiology reports, laboratory reports, or other patient records. The NCI C3D will serve as the primary source from which all research analyses will be performed.  
6.1.5.2 Each subject's record will be reviewed for verification of eligibility by the research nurse, transplant coordinator, and P.I. or authorized designee prior to consent signing. Documentation of this review will be made on the eligibility checklist and a copy kept in each subject's research record. The research nurse will verify enrollment data entered into the C3D database for each subject within 4 weeks of signing consent. Ongoing data entered into C3D will be verified by the research nurse at completion of induction chemotherapy, after day 28 and day 100 post transplant then bi-annually prior to each continuing review submission. Documentation of data verification will be tracked in the C3D database. Also no data will be collected beyond 5 years except for survival.  
6.1.5.3 Data will also be sent to the International Bone Marrow Transplant Registry
(IBMTR).
6.1.5.4 Additional baseline and follow-up data may be requested by the NMDP which are not required for this protocol. Recipients will be offered the opportunity to participate in a cross-institute, NIH NMDP Research Data Collection protocol available to all recipients of NMDP hematopoietic stem cell products for which consent is optional. This optional data collection protocol ensures compliance with standards described in the Transplant Center Agreement between the NIH Clinical Center and the NMDP.  
All data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant.  
All adverse events, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event. Patients will be followed for adverse events for a minimum of 30 days after the last administration of investigational agent/intervention and have an attribution of at least possibly related to the agent/intervention should be recorded and reported as per section 8.2 or until removal from study treatment, whichever comes first.  
An abnormal laboratory value will be considered an AE if the laboratory abnormality is characterized by any of the following:
- Results in discontinuation from the study  
- Is associated with clinical signs or symptoms
- Requires treatment or any other therapeutic intervention
- Is associated with death or another serious adverse event, including hospitalization.  
- Is judged by the Investigator to be of significant clinical impact
- If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient's outcome.
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH
Intramural Records Retention Schedule as applicable.  
Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, the IRB will be notified. 6.1.6 Long term follow-up guidelines Patients may obtain these studies within 2 months of desired date and these may be done in other institutions than NIH with documentation. Please note that these should be performed yearly after transplantation either as part of this protocol or in another setting.  
a) Dental evaluations day +365  
b) TSH, Ferritin and iron studies on day +180, +365, 24 months, 3, 4, 5 years c) FSH, LH, estradiol, on day +365 and 24 months (females only) d) Ophthalmological evaluation on day +365 e) DEXA scan on day +365, then yearly if abnormal or on steroids f) Echocardiogram on day +100, then only if clinically indicated g) GYN examination (female patients) day +365 h) Urinalysis/protein screening day +180, +365 i) Pulmonary Function Test on day +100; and at 6, 9, 12, 18, 24 months and then annually post-transplant thereafter up until the 5 year timepoint.  
6.2 RESPONSE CRITERIA 6.2.1 Complete Response (CR):
6.2.1.1 Non-Hodgkin's lymphoma or Hodgkin's lymphoma: complete disappearance of all detectable signs and symptoms of lymphoma for a period of at least one month. All lymph nodes and nodal masses must have regressed to normal size (< 1.5 cm in greatest transverse diameter for nodes > 1.5 cm before therapy). Previously involved nodes that were 1.1 to 1.5 cm in greatest transverse diameter before treatment must have decreased to < 1 cm in their greatest transverse diameter after treatment or by more than 75% in the sum of the products of the greatest diameters (SPD). The spleen (or any other organ), if considered to be enlarged due to involvement by lymphoma before therapy and on the basis of a CT scan, must have regressed in size and must not be palpable on physical examination. Any macroscopic nodules in any organs detectable on imaging techniques should no longer be present. If bone marrow was involved by lymphoma before treatment, the infiltrate must be cleared on repeat bone marrow aspirate and biopsy of the same site [104]. Response criteria may be modified upon publication of revised response criteria [109].  
6.2.1.2 Chronic lymphocytic leukemia: complete resolution of all detectable signs and symptoms for at least 2 months, with peripheral blood lymphocytes < 4K/l, neutrophils > 1.5K/l, platelets > 100K/l, hemoglobin > 11g/dl
(untransfused), bone marrow lymphocytes < 30% without lymphoid nodules.  
6.2.1.3 Chronic myelogenous leukemia: Hematologic CR - normalization of peripheral blood counts (WBC < 10K/l, platelets < 450K/l), no immature cells on peripheral smear (blasts, promyelocytes, metamyelocytes). Cytogenetic CR - hematologic CR, with cytogenetic studies negative for Philadelphia chromosome (Ph). Molecular CR - hematologic and cytogenetic CR, with PCR studies negative for *bcr-abl*.  
6.2.1.4 Other leukemias, myeloproliferative disorders, or myelodysplastic syndrome:
hematologic remission is defined as normalization of peripheral blood counts
(ANC > 1,500/μl and platelets > 100,000/mm3) without circulating blasts, bone marrow cellularity > 20% with normal maturation, fewer than 5% blasts in bone marrow; and no detectable Auer rods [106]. Extramedullary leukemia may not be present. The absence of specific molecular or cytogenetic markers of disease that were previously present further defines molecular or cytogenetic remission, respectively. In the case of myeloproliferative disorders, there must be absence of bone marrow fibrosis with normal hematologic parameters.  
6.2.1.5 Multiple myeloma [111]: absence of urine and serum M-components by immunofixation and electrophoresis; normalization of immunoglobulins is not required. Clonal plasma cells in bone marrow <1% by PCR or flow cytometry. Normal serum calcium. No new bone lesions; no enlargement of existing lesions. Vertebral collapse or other pathological fracture due to osteoporosis or existing lesion does not prevent categorization as CR.
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6.2.2 complete response/unconfirmed (cru)

6.2.2.1 Non-Hodgkin's lymphoma or Hodgkin's lymphoma: A residual lymph node mass > 1.5 cm in greatest transverse diameter will be considered CRu if it has
regressed by more than 75% in the SPD, does not change over at least one month, is negative by PET or gallium, and is negative on any biopsies obtained (biopsy not required). Individual nodes that were previously confluent must have regressed by more than 75% in their SPD compared with the size of the original mass and be stable for at least one month. Any residual lesions in involved organs must have decreased by more than 75% in the SPD or be < 1 cm, be clinically consistent with residual scarring, and be stable for at least one month. Indeterminate bone marrow, if previously involved with lymphoma, will also be considered CRu.
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6.2.3 partial response (pr)

6.2.3.1 Non-Hodgkin's lymphoma or Hodgkin's lymphoma: a 50% or greater decrease in SPD of all measured lesions lasting for a period of at least one month. No individual lesion may increase in size, and no new lesions may appear [108].  
6.2.3.2 Chronic lymphocytic leukemia: a 50% or greater decrease in SPD of measured lymph nodes, hepatomegaly, or splenomegaly lasting at least 2 months, plus one or more of neutrophils > 1.5K/l, platelets > 100K/l, or hemoglobin >
11g/dl (or 50% improvement).
6.2.3.3 Chronic myelogenous leukemia: Hematologic PR - as for hematologic CR,
except for (1) persistence of immature cells, or (2) platelets < 50% pretreatment level but > 450K/l, or (3) persistent splenomegaly but > 50% of pretreatment size. Cytogenetic PR - hematologic CR, with 1-34% Ph-positive cells (major response). Cytogenetic minor response - hematologic CR, with 35-90% Phpositive cells.  
6.2.3.4 Acute leukemias, MDS or myeloproliferative disorders in transformation: All criteria for complete remission are satisfied (Section **6.2.1**), except that the bone marrow may contain > 5% but < 25% blasts, or < 5% blasts are present with Auer rods or abnormal morphology.  
6.2.3.5 Multiple myeloma:
a) Very good partial response - reduction by > 75% in serum myeloma protein production, with decrease in Bence-Jones proteinuria by > 90%. Clonal marrow plasmacytosis < 5%. No new lytic bone lesions.  
b) Partial response - Reduction by > 50% in serum myeloma protein production maintained for a minimum of six weeks.
6.2.4 Stable Disease (SD):
6.2.4.1 Non-Hodgkin's lymphoma or Hodgkin's lymphoma: tumor measurements not meeting the criteria for CR, CRu, PR, or PD.  
6.2.4.2 Chronic lymphocytic leukemia: response parameters not meeting criteria for CR, PR, or PD.
6.2.5 Relapsed or Progressive Disease (PD):
6.2.5.1 Non-Hodgkin's lymphoma or Hodgkin's lymphoma: a 50% or greater increase in SPD of all measured lesions compared to the smallest previous measurements, or appearance of any new lesion(s).  
6.2.5.2 Chronic lymphocytic leukemia: a 50% or greater increase in SPD of all measured lesions compared to the smallest previous measurements, or appearance of any new lesion(s); a > 50% increase in circulating lymphocytes; Richter's syndrome.  
6.2.5.3 Chronic myelogenous leukemia: Increase in the number of metaphases demonstrating Ph by cytogenetics or t(9:22) by FISH; return to PCR positivity for *bcr-abl* after previously becoming negative.  
6.2.5.4 Other leukemias, myeloproliferative disorders, or myelodysplastic syndrome:
bone marrow and peripheral blood morphological features consistent with relapse or progression, including rising blast count and re-emergence of specific molecular or cytogenetic markers.
6.2.5.5 Multiple myeloma (requires 2 of the following): an increase in serum Mprotein to > 50% above the lowest level or a rise > 2 g/dl; an increase in urine light chain excretion to 50% above the lowest value (at least 250 mg/24 hours) or an increase > 2 g/24 hours of light chain excretion; an increase in soft tissue plasmacytomas by 50% or new or increasing lytic bone lesions; the above protein criteria for relapse, plus hypercalcemia > 12 mg/dl, anemia with hemoglobin decrease > 2 g/dl, increased bone marrow plasma cells by 50%, or generalized bone pain.
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6.3 toxicity criteria

6.3.1 The NCI Common Terminology Criteria for Adverse Events Version 3.0 (CTCAEv3, available via NCI web site (http://ctep.cancer.gov/forms/CTCAEv3.pdf) will be utilized for evaluation of toxicity.  
6.3.2 Acute and chronic graft-versus-host disease (GVHD) will be graded according to criteria specified in Appendix C.  
6.4 STATISTICAL CONSIDERATIONS
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6.4.1 primary objectives

6.4.1.1 Determination of CD4+ T cell receptor Vβ repertoire - A primary objective of this study is to conduct a pilot trial to determine the effects of two biologically distinct GVHD prophylaxis regimens (tacrolimus/methotrexate/sirolimus [TMS] and alemtuzumab/ cyclosporine [AC]) on immune reconstitution in patients receiving targeted-immune depletion and reduced-intensity allogeneic HSCT from HLA-matched unrelated donors. As part of a comprehensive assessment of immune reconstitution, the study will primarily focus on differences in CD4+ T cell receptor Vβ repertoire and try to determine in a pilot setting if a) use of either of the two regimens in this trial will have a substantial impact on this parameter at three or 12 months post-transplant, and b) if, again within the context of a pilot study, if there is a difference between the two arms with respect to the magnitude of the change from baseline in this parameter at 3 and 12 months. These will data will be complemented by additional analyses on immune reconstitution which include determination of CD4+ T cell receptor Vβ repertoire at other time points (1, 3, 6, and 12 months), changes in CD8+ T cell receptor Vβ repertoire at 1, 3, 6 and 12 months, the kinetics of CD4+ and CD8+ T-cells and NK cell depletion and recovery, IL-7 and IL-15 cytokine levels, and monocyte production of inflammatory cytokines, as part of the secondary objectives.  
6.4.1.1.1 This trial was initially designeded to be a pilot study, with patients stratified according to whether they have 7/8 or 8/8 matches. To meet the primary objective of immune reconstitution, a total of 50 patients with 8/8 matches will undergo transplantation, 25 per arm, and be randomized to receive either an alemtuzumab-based prophylaxis (AC) or triple agent prophylaxis
(TMS) based on the order they enrolled onto the study. In addition, 26 patients with 7/8 matches, 13 per arm, will also be randomized to undergo transplantation, and will be evaluated separately. In the situation where a patient is assigned to an arm which is temporarily closed as it is being assessed for toxicities relative to early stopping rules (section **6.4.3**), the patient will be randomized to the arm for which accrual is open. The primary evaluation, for which this number was selected, will be with respect to the change in the CD4+ T cell repertoire diversity, as determined by spectratype complexity index, at 3 and 12 months post-transplant as compared to normal healthy donors. The recipient spectratype complexity index within a Vβ family will be determined as a percentage of the number of peaks in its spectratype histogram compared to the number of peaks in the corresponding donor Vβ family spectratype histogram [49]. Any Vβ family with a complexity index of ≥85% was considered to be fully complex. Histogram peaks will be defined by the Genotyper Genescan software analysis program.  
6.4.1.1.2 The study was initially designed to allow for 10 patients per arm, in order to have 91% power to detect a difference between the arms that is equal to 1.5 standard deviations of the intra-arm difference in the single primary parameter, spectratype complexity index at 3 months, with a 0.10 two-sided alpha level test. It will be assumed that the comparison will be made with a two-sample t-test, but if the data in either arm are not normally distributed (p<0.05 by a Shapiro-Wilks test), then a Wilcoxon rank sum test will be used. Amendment F will increase the size of the study in order to enroll 20 evaluable patients per arm who have 8/8 matches. With 20 patients per arm who have no mismatches, there will be 86% power to detect a difference between the two arms equal to 1.0 standard deviations of the intra-arm difference of the 3 month and 12 month CD4+ T cell repertoire diversity measures, with a 0.05 two-sided alpha level test. It will be assumed that the comparisons will be made with a two-sample t-test. However, if the data on either arm is not normally distributed (p<0.05 by a Shapiro-Wilks test), then a Wilcoxon rank sum test will be used. The 20 evaluable patients (10/arm) from among 26 transplanted who have 7/8 matches will have results compared between the two arms using the same criteria as in the original protocol design.  
6.4.1.1.3 Secondly, the difference in CD4+ T cell repertoire diversity between baseline and 3 months, and baseline and 12 months will be made using a paired t-test if the data are normally distributed. The study originally noted that with 10 patients on a given arm, there is 80% power to detect a 1 SD effect size with a two-sided original design, although there are two arms with this comparison being made within each arm, no formal correction for the two comparisons was required. If the data are not normally distributed, then a Wilcoxon signed rank test would be used. With Amendment F, with 20 evaluable patients per arm from among 25 transplanted, there is 80%
power to detect a 0.75 SD effect size for each of the two endpoints within an arm, with a two-sided 0.025 alpha level test for each evaluation. This will allow for an overall 0.05 level test for the two comparisons within each arm, assuming that a Bonferroni adjustment is used. Because of the pilot nature of the original design, although there are two arms with these comparisons being made within each arm, no formal correction for the two comparisons will be required. If the data are not normally distributed, then a Wilcoxon signed rank test will be used. The 20 evaluable patients (10/arm) from among 26 transplanted who have 7/8 matches will have results compared within each arm using the same criteria as in the original protocol design. In order to allow for up to 20% of patients who may die prior to the time of the treatment evaluation point, accrual of up to 25 patients per arm will be permitted for the patients with 8/8 matches and 13/arm for the 7/8 matches in order to yield 20/arm and 10/arm who are evaluable for the two strata.  
6.4.2 Determine and monitor incidence, organ severity and overall severity of chronic GVHD -
As stated above (section **6.4.1.1.1**) the study was originally intended as a pilot, but it was expanded upon meeting initial safety criteria to 50 patients with 8/8 HLA-matched donors and to study patients with 7/8 HLA-matched donors in a pilot manner. Results from this expanded trial indicate that there is an opportunity to study chronic GVHD in a detailed manner (please refer to section 1.5). Specifically it is a primary arm of this study to determine the incidence of severe chronic using the NIH Consensus Conference diagnosis and staging criteria.  
6.4.2.1 Patients will continue to be randomized between these two arms as they have been prior to the amendment. Patients will continue to be stratified according to the degree of matching: 7/8 vs. 8/8 matches. This portion of the trial will be conducted in order to obtain modestly precise estimates of the rates of chronic GVHD in patients treated on either AC or TMS so that we may learn more about the effect of AC vs. TMS on this disease and to permit development of a subsequent, more definitive trial in this patient population. As such, patients enrolled on this trial prior to this amendment will be included in the total patients evaluated with respect to this endpoint.  
6.4.2.2 Based on data prior to the initiation of this amendment, is has been estimated that approximately 20% of the patients on each arm will eventually develop severe chronic GVHD. If there were 44 evaluable patients on each arm who survive beyond 100 days post transplant, this would allow a 90% two-sided confidence interval around the observed proportion to have a width of +/-10% from the observed proportion when the expected proportion of patients with severe cGVHD is estimated to be 20%.  
6.4.2.3 It is anticipated that 90% of all patients enrolled will be evaluable for evaluation of chronic GVHD in that they survive 100 days post transplant or longer. Thus, in order to enroll 88 total evaluable patients, it is expected that approximately 100 would be needed. If 105 were enrolled and if the true fraction surviving to 100 days was 90%, the probability that 88 or more would survive beyond 100 days is 98.3%; if 100 were enrolled, the probability that 88 or more would survive beyond 100 days is 80%. As such, we will plan to set an accrual ceiling at 105 in order to assure accrual of 88 evaluable patients. Approximately 3-5 patients per month are expected to be able to be randomized to this trial if one or two institutions are participating. Thus, with approximately 50 of the 105 already enrolled, it is anticipated that an additional 1-2 years may be required to complete the enrollment of the 105 randomized patients.
6.4.3 In addition, it will be important to determine the overall safety of both of these regimens in the setting of targeted sequential immune-depleting chemotherapy followed by reduced-intensity allogeneic HSCT from HLA-matched unrelated donors, separately for the patients with 8/8 matches and 7/8 matches. An evaluation of engraftment, severe acute GVHD, early and late treatment-related mortality and overall survival will be performed relative to each arm of this trial, for the 7/8 match and 8/8 match strata separately:
6.4.3.1 Graft Rejection - Early stopping for graft rejection based on CD3 chimerism will also be implemented. Any patient for whom the percentage of CD3 cells is <10% at day 100 will be considered to have rejected the graft. A maximum of 15% of patients with graft rejection is the greatest fraction tolerable.  
Patients will continue to be accrued to a specific arm provided that no greater than 2 patients in the 10 within that arm have rejected their graft (and no other stopping rules are implemented). The following table, based on binomial probability calculations, shows the probability of having 2 or more patients rejecting their grafts by 10 patients, for a set of possible underlying true probabilities.  
| Probability of   | Probability of early termination   |
|------------------|------------------------------------|
| graft Rejection  | due to graft rejection             |
| .05              | .09                                |
| .10              | .26                                |  
.$46\,$  .
.$15\,$  .
.15 .46
.$62\,$  .
$\mathbf{M}$
.20 .62
.25 .72 Thus, if the true probability of graft rejection is about 15%, there is a 46% probability that 2 or more patients will have this occur within the 10 patients on a specific arm and thus need to end accrual to this arm as soon as this can be determined.  
6.4.3.2 Acute GVHD - Acute GVHD will be determined actuarially. Kaplan-Meier curves will be constructed for each arm separately. Acute GVHD will be determined by standard criteria [106]. Stopping rules for grade III or IV acute GVHD will also be implemented. It has been noted that 20% grade III or IV acute GVHD is the upper limit for an acceptable rate, so a stopping rule based on exceeding this rate will be implemented.
Patients will continue to be accrued to a specific arm provided that no greater than 2 patients in the 10 within that arm have developed grade III or IV acute GVHD (and no other stopping rules are implemented). The following table, based on binomial probability calculations, shows the probability of having 2 or more patients develop grade III or IV acute GVHD by 10 patients, for a set of possible underlying true probabilities.  
| Probability of grade   | Probability of early termination   |
|------------------------|------------------------------------|
| III or IV acute GVHD   | due to grade III or IV acute GVHD  |
| .05                    | .09                                |
| .10                    | .26                                |
| .15                    | .46                                |
| .20                    | .62                                |
| .25                    | .72                                |  
Thus, if the true probability of grade III or IV acute GVHD is about 20%, there is a 62% probability that 2 or more patients will have this occur within the 10 patients on a specific arm and thus need to end accrual to this arm as soon as this can be determined.  
6.4.3.3 Early treatment-related mortality - It will be important to continuously monitor the treatment-related mortality, and to have a stopping rule in the event that this is excessive. It has been noted that 20% 100-day treatment-related mortality is the upper limit for an acceptable rate, so a stopping rule based on attaining this rate or exceeding or rate will be implemented, separately for each arm. Treatment-related mortality (TRM) is defined as any death occurring within 28
days after transplantation and any death occurring 28 days or more after transplantation in a patient in continuous remission.  
Patients will continue to be accrued to a specific arm provided that no greater than 2 patients in the 10 within that arm have died by day 100 due to treatment-related causes (and no other stopping rules are implemented). The following table, based on binomial probability calculations, shows the probability of having 2 or more patients die from treatment-related causes by 10 patients, for a set of possible underlying true probabilities.  
| Probability of   | Probability of early termination   |
|------------------|------------------------------------|
| 100 day TRM      | due to 100 day TRM                 |
| .05              | .09                                |
| .10              | .26                                |
| .15              | .46                                |
| .20              | .62                                |
| .25              | .72                                |  
Thus, if the true probability of day 100 TRM is about 20%, there is a 62% probability that 2 or more patients will have this occur within the 10 patients on a specific arm and thus need to end accrual to this arm as soon as this can be determined.  
6.4.3.4 Late mortality - One year treatment-related mortality and one year overall survival will also be evaluated and used to determine accrual may proceed to a given arm. If greater than approximately 33% of patients are determined to have died due to treatment by one year on a specific arm, then that arm of the trial as originally designed will no longer accrue patients.  
6.4.3.5 Late treatment-related mortality - Accrual to the particular arm of the trial will stop if 2 or more patients in the first 6 with that arm have treatment-related mortality within the first year, or this has not been met, if 3 or more in the 10 with that arm have TRM within the first year. The following table, based on binomial probability calculations, shows the probability of having 2 or more patients in the first 6 dying from TRM in the first year, or 3 or more of the first 10 dying from TRM in the first year, for a set of possible underlying true probabilities.
Probability of 1 year treatment-related mortality Probability of early termination due to 1 year treatment-related mortality  
.1 .07 .2 .33  
.35 .73
.4 .82 .5 .94 Thus, if the true probability of one year TRM is 35%, there is a 73% probability that this rule will be invoked within the 10 patients on a specific arm and thus need to end accrual to this arm as soon as this can be determined.  
6.4.3.6 Overall Survival - Monitoring of overall survival will be performed and if 50%
or greater patients within 10 or fewer enrolled on a specific arm are ever determined to not survive until one year for whatever reason, then that arm will no longer accrue patients as it was originally designed.  
Accrual to the particular arm of the trial will stop if 3 or more patients in the first 6 within that arm have died from any cause within the first year, or this has not been met, if 5 or more in the 10 in that arm have died from any cause within the first year. The following table, based on binomial probability calculations, shows the probability of having 3 or more patients in the first 6 on a specific arm dying from any cause in the first year, or 5 or more of the 10 in that arm dying from any cause in the first year, for a set of possible underlying true probabilities.  
| Probability of 1   | Probability of early termination   |
|--------------------|------------------------------------|
| year mortality     | due to 1 year mortality            |
| .3                 | .16                                |
| .4                 | .39                                |
| .5                 | .64                                |
| .6                 | .84                                |  
6.4.3.7 Although ideally, there would be no additional patients accrued pending determination of whether a specific stopping rule must be invoked for a specific arm, to allow continuity of patients being referred into the trial, up to three additional patients may be permitted to be accrued beyond that needed to evaluate the particular stopping rule.  
6.4.3.8 It should be noted for any of the above monitoring plans, that observation of the maximum number of events in 10 patients within a specific arm means that subsequent study of this arm, as specified on this protocol, is inadvisable from a safety perspective, although technically the arm was allowed to continue until the full 10 patient accrual goal within an arm has been met.  
6.4.3.9 If one arm of the study is temporarily or permanently closed, then accrual will be permitted to the remaining open arm.  
6.4.4 *Secondary objectives* - As secondary endpoints, additional immune reconstitution parameters will be evaluated at multiple time points in an exploratory fashion within the context of a pilot study. Since these will be considered secondary endpoints, no formal adjustment for multiple comparisons will be undertaken, and the comparisons will be reported in the context of a hypothesis generating setting.  
6.4.4.1 It is anticipated that approximately 1-2 patients per month may be enrolled onto this study. Thus, to accrue a total of 60 evaluable patients to both arms (up to 25 per arm for the patients with no mismatches and 13 per arm for the arms with 7/8 matches) it is anticipated that up to 3 years will be required. To allow for the possibility of a small number of patients who may be inevaluable (e.g. disease progression prior to transplant), the accrual ceiling will be set at 85 patients or 76 patients who receive a transplant, whichever comes first.
6.4.5 There are no gender or racial/ethnic restrictions on patient selection. This protocol is open to both genders and all racial/ethnic groups.
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7 human subject protections 7.1

Patients with high-risk hematologic malignancies will be the subjects for this study. Allogeneic HSCT represents a potentially curative treatment for patients with the disease characteristics selected for inclusion (section **2.1.1**). The expected survival of such patients with conventional chemotherapy is approximately one year or shorter. Eligibility in this protocol is limited to adults (age > 18-74 years). 7.2 PARTICIPATION OF CHILDREN Children will not be enrolled in this study. Immune reconstitution is different between children and adults, which is the primary endpoint of this study. 7.3 PARTICIPATION OF NIH SUBJECTS UNABLE TO GIVE CONSENT Adults unable to give consent are excluded from enrolling in the protocol. However re-consent may be necessary and there is a possibility, though unlikely, that subjects could become decisionally impaired. For this reason and because there is a prospect of direct benefit from research participation (section 7.4), all subjects ≥ age 18 will be offered the opportunity to fill in their wishes for research and care, and assign a substitute decision maker on the "NIH Advance Directive for Health Care and Medical Research Participation" form so that another person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH Ability to Consent Assessment Team (ACAT) for evaluation as needed for the following: an independent assessment of whether an individual has the capacity to provide consent; assistance in identifying and assessing an appropriate surrogate when indicated; and/or an assessment of the capacity to appoint a surrogate. For those subjects that become incapacitated and do not have pre-determined substitute decision maker, the procedures described in MEC Policy 87-4 and NIH HRPP SOP 14E for appointing a surrogate decision maker for adult subjects who are (a) decisionally impaired, and (b) who do not have a legal guardian or durable power of attorney, will be followed.
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7.4 risks/benefits analysis

There is a potential benefit to the transplant recipient, as demonstrated by the published literature documenting long-term disease-free survival for patients with high-risk hematologic malignancies following allogeneic HSCT. This carefully defined patient population is a reasonable one in which to explore strategies to improve the results of allogeneic HSCT from unrelated donors, which historically has achieved prolonged remission and survival for some patients but has been associated with significant morbidity and high transplant-related mortality. The strategies chosen in this protocol extend our previous work to develop a platform for consistent engraftment of allogeneic cells through immunoablative induction chemotherapy, host T cell depletion, and reduced-intensity conditioning. Studying this platform in the setting of allogeneic HSCT from unrelated donors is a logical, planned extension of our previous efforts. The potential risks to the transplant recipient have been carefully considered and are felt to be potentially less than would otherwise occur with myeloablative allogeneic HSCT. The risks graft rejection, acute and chronic GVHD, infection, and relapse will be present, as discussed previously. 7.5 INFORMED CONSENT PROCESS AND DOCUMENTATION The procedures and treatments involved in this protocol, with their attendant risks and discomforts, potential benefits, and potential alternative therapies will be carefully explained to the recipient. Similarly, the procedures and treatments involved in this protocol, with their attendant risks and discomforts, will be carefully explained to the donor at the respective donor center as required by NMDP. A signed informed consent document will be obtained from both the recipient and the donor. Consent forms: The original signed consent goes to Medical Records; a copy will be placed in research record, as per NIH policy, and a copy will be kept with the patient's chart. A copy of the informed consent documents will also be given to the recipient. The Clinical Coordinator, Data Management Section, will ascertain the date of IRB approval before registering the first patient. The informed consent document contains all elements required for consent. In addition, the Principal Investigatoror their designee will obtain oral consent and will be available to answer all patient questions. 7.5.1 Telephone re-consent procedure Re-consent on this study may be obtained via telephone according to the following procedure: The informed consent document will be sent to the subject. An explanation of the study will be provided over the telephone after the subject has had the opportunity to read the consent form. The subject will sign and date the informed consent. A witness to the subject's signature will sign and date the consent. The original informed consent document will be sent back to the consenting investigator who will sign and date the consent form with the date the consent was obtained via telephone. A fully executed copy will be returned via mail for the subject's records. The informed consent process will be documented on a progress note by the consenting investigator.
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8 safety reporting requirements/data and

Any untoward medical occurrence in a human subject, including any abnormal sign (for example, abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with the subject's participation in research, whether or not considered related to the subject's participation in the research.
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8.1.2 suspected adverse reaction

Suspected adverse reaction means any adverse event for which there is a reasonable possibility that the drug caused the adverse event. For the purposes of IND safety reporting, 'reasonable possibility' means there is evidence to suggest a causal relationship between the drug and the adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality than adverse reaction, which means any adverse event caused by a drug.
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8.1.3 unexpected adverse reaction

An adverse event or suspected adverse reaction is considered "unexpected" if it is not listed in the investigator brochure or is not listed at the specificity or severity that has been observed; or, if an investigator brochure is not required or available, is not consistent with the risk information described in the general investigational plan or elsewhere in the current application. "Unexpected", also refers to adverse events or suspected adverse reactions that are mentioned in the investigator brochure as occurring with a class of drugs or as anticipated from the pharmacological properties of the drug, but are not specifically mentioned as occurring with the particular drug under investigation.
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8.1.4 serious

An Unanticipated Problem or Protocol Deviation is serious if it meets the definition of a Serious Adverse Event or if it compromises the safety, welfare or rights of subjects or others. 8.1.5 Serious Adverse Event An adverse event or suspected adverse reaction is considered serious if in the view of the investigator or the sponsor, it results in any of the following:
- Death,  
- A life-threatening adverse drug experience
- Inpatient hospitalization or prolongation of existing hospitalization - Persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions  
- A congenital anomaly/birth defect.  
- Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.  
8.1.6 Disability
A substantial disruption of a person's ability to conduct normal life functions.  
8.1.7 Life-threatening adverse drug experience Any adverse event or suspected adverse reaction that places the patient or subject, in the view of the investigator or sponsor, at immediate risk of death from the reaction as it occurred, i.e., it does not include a reaction that had it occurred in a more severe form, might have caused death. 8.1.8 Protocol Deviation (NIH Definition) Any change, divergence, or departure from the IRB approved research protocol. 8.1.9 Non-compliance (NIH Definition) The failure to comply with applicable NIH Human Research Protections Program (HRPP) policies, IRB requirements, or regulatory requirements for the protection of human research subjects. 8.1.10 Unanticipated Problem Any incident, experience, or outcome that:
- Is unexpected in terms of nature, severity, or frequency in relation to  
a) the research risks that are described in the IRB-approved research protocol and informed consent document; Investigator's Brochure or other study documents, and b) the characteristics of the subject population being studied; AND
- Is related or possibly related to participation in the research; AND - Suggests that the research places subjects or others at a g*reater risk of harm* (including physical, psychological, economic, or social harm) than was previously known or recognized.  
8.1.11 MedWatch Reporting
Serious adverse events that occur with commercially available agents will be reported directly to the FDA via Medwatch (http://www.fda.gov/medwatch). 8.2 NCI-IRB AND CLINICAL DIRECTOR REPORTING 8.2.1 NCI-IRB and NCI Clinical Director Expedited Reporting of Unanticipated Problems and Deaths The Protocol PI will report in an NIH Problem Form to the NCI-IRB and the NCI Clinical Director:
- All deaths, except deaths due to progressive disease  
- All Protocol Deviations
- All Unanticipated Problems
- All non-compliance Reports must be received within 7 days of PI awareness via iRIS. 8.2.2 NCI-IRB Requirements for PI Reporting at Continuing Review The protocol PI will report to the NCI-IRB:  
1. A summary of all protocol deviations in a tabular format to include the date the deviation occurred, a brief description of the deviation and any corrective action.  
2. A summary of any instances of non-compliance 3. A tabular summary of the following adverse events:
- All Grade 2 unexpected events that are possibly, probably or definitely related to the research;
- All Grade 3 and 4 events that are possibly, probably or definitely related to the research;
- All Grade 5 events regardless of attribution;
- All Serious Events regardless of attribution.  
NOTE: Grade 1 events are not required to be reported.
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8.3 data safety and monitoring

The clinical research team will meet on a weekly basis when patients are being actively treated on the trial to discuss each patient. Decisions about dose level enrollment and dose escalation if applicable will be made based on the toxicity data from prior patients. All data will be collected in a timely manner and reviewed by the principal investigator or a lead associate investigator. Adverse events will be reported as required above. Any safety concerns, new information that might affect either the ethical and or scientific conduct of the trial, or protocol deviations will be immediately reported to the IRB using iRIS. The principal investigator will review adverse event and response data on each patient to ensure safety and data accuracy. The principal investigator will personally conduct or supervise the investigation and provide appropriate delegation of responsibilities to other members of the research staff.
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8.3.2 safety monitoring committee (smc)

This protocol will require oversight from the Safety Monitoring Committee (SMC). Initial review will occur as soon as possible after the annual NCI-IRB continuing review date. Subsequently, each protocol will be reviewed as close to annually as the quarterly meeting schedule permits or more frequently as may be required by the SMC. For initial and subsequent reviews, protocols will not be reviewed if there is no accrual within the review period. Written outcome letters will be generated in response to the monitoring activities and submitted to the Principal investigator and Clinical Director or Deputy Clinical Director, CCR, NCI. 8.4 RECORD KEEPING 8.4.1 All patients must have signed an Informed Consent, and on-study eligibility checklist will be filled out by the Research RN and faxed to the Central Registration Office (CRO) before patient is entered on the study.  
8.4.2 Complete records must be maintained on patients; these will consist of the hospital chart with any supplementary information obtained from outside laboratories, radiology reports, or physician's records. These records will serve as the primary source material that forms the basis for the research record. All relevant data will also be entered on the NCI C3D database from which formal analyses are done. The primary source documentation will assure the following: on-study information, including patient eligibility data and patient history; flowsheets, specialty forms for pathology, radiation, or surgery; adverse event assessment; and off-study summary sheet, including a final assessment by the treating physician.  
8.4.3 Paper CRFs (see **Appendix** K) will be used to aid in data collection on this study.
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9 pharmaceutical information 9.1 cyclophosphamide

9.1.1 Supply - Cyclophosphamide will be purchased by the NIH Clinical Center Pharmacy Department from commercial sources and is supplied as a lyophilized powder in various vial sizes.  
9.1.2 Preparation - will be reconstituted with sterile water for injection to yield a final concentration of 20 mg/ml as described in the package insert.  
9.1.3 Storage and Stability - The vials are stored at room temperature. Following reconstitution as directed, solutions of cyclophosphamide are stable for 24 hours at room temperature, or 6 days when refrigerated at 2-8 C.  
9.1.4 Route of Administration - The cyclophosphamide used in this regimen will be mixed in 100 ml 0.9% sodium chloride, Inj., and given as an IVPB over 30 minutes in the induction regimen (i.e. EPOCH-F/R; Section **3.5.3**). In the preparative regimen (Section 3.6.5), it is given over two hours.
9.1.5 Toxicities  
a) Nausea and vomiting - variable; symptomatically improved with standard anti-emetics and/or benzodiazepines [e.g., lorazepam].  
b) Water retention - cyclophosphamide may rarely provoke the syndrome of inappropriate antidiuretic hormone secretion and resultant hyponatremia, usually manifested 12-48 hrs after IV administration, necessitating frequent accurate assessment [q 1-2 hrs] of intake, urine output and urine specific gravity. This effect can be counteracted by furosemide. Fluid restriction is not feasible during administration of high dose cyclophosphamide.  
c) Cardiomyopathy - cyclophosphamide may cause severe, sometimes lethal, hemorrhagic myocardial necrosis or congestive cardiomyopathy. Patients may present with congestive cardiomyopathy as late as 2 weeks after the last dose of cyclophosphamide. The clinical syndrome has been observed in patients receiving the dose of cyclophosphamide used in this protocol. In an attempt to minimize this complication, patients with significant cardiac dysfunction are excluded from this protocol [see patient eligibility]. Congestive failure is managed according to standard medical therapeutics.  
d) Hemorrhagic cystitis - this is a serious, potentially life-threatening complication related to injury of the bladder epithelium by cyclophosphamide metabolites. Although subclinical hematuria is not uncommon at this dose level, clinically significant hematuria or serious hemorrhage can usually be avoided by maintaining a high urine volume and frequent voidings and the administration of Mesna. Diuresis is maintained for 24 hrs after completion of last dose by parenteral infusions of normal saline with potassium chloride, as specified in section **3.6.6**. Careful monitoring of serum and urine electrolytes is mandated. Furosemide may be required to ensure this diuresis. Continuous bladder irrigation may be used for control of significant hematuria.  
e) Sterility f) Less common but serious complications include pulmonary fibrosis and secondary malignancies. Less common but reversible toxicities include alopecia and skin rash.  
9.2 MESNA (SODIUM 2-MERCAPTOETHANESULFONATE, MESNUM, MESNEX, NSC-113891)
9.2.1 Supply - Mesna will be will be purchased by the NIH Clinical Center Pharmacy Department from commercial sources and is supplied as a 100mg/ml solution.  
9.2.2 Preparation - Dilute up to 20 mg Mesna/ml fluid in D5W or 0.9% sodium chloride, Inj.  
Mesna should be started concurrently with the cyclophosphamide in the preparative regimen (Section 3.4.3). Mesna will be given at 1200 mg/m2in 500 ml by continuous IV
infusion over 24 hour infusion for four doses (days -6, -5, -4, and -3).  
9.2.3 Storage and Stability - Intact ampules are stored at room temperature. Diluted solutions
(1 to 20 mg/dl) are physically and chemically stable for at least 24 hours under refrigeration. Mesna is chemically stable at room temperature for 48-72 hours in D5W, 48-72 hour in D5W/0.45% sodium chloride injection, or 24 hours in 0.9% sodium chloride injection.  
9.2.4 Administration - To be administered intravenously as continuous infusion. 9.2.5 Toxicities - Nausea, vomiting, diarrhea.
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9.3 filgrastim (g-csf neupogen)

9.3.1 Supply - Commercially available as filgrastim injection in a concentration of 300g/ml in 1ml (300g) and 1.6ml (480g) vials.  
9.3.2 Preparation - For subcutaneous administration, the appropriate prescribed dose is drawn up from the vial with no further dilution prior to administration. For intravenous administration, the commercial solution for injection should be diluted prior to administration. It is recommended that the prescribed dose be diluted with dextrose 5%
in water to a concentration greater than 5g/ml. Dilution of filgrastim to a final concentration of less than 5g/ml is not recommended at any time. Do not dilute with saline at any time; product may precipitate. Filgrastim diluted to concentrations between 5 and 15g/ml should be protected from absorption to plastic materials by the addition of Albumin (Human) to a final concentration of 2mg/ml. When diluted in 5% dextrose or 5% dextrose plus Albumin (Human), filgrastim is compatible with glass bottles, PVC and polyolefin IV bags, and polypropylene syringes. The dose may be "rounded down" to within 10% of patient's calculated dose to use the drug cost-effectively.  
9.3.3 Storage and Stability - Filgrastim for injection should be stored in the refrigerator at 2 oto 8 oC (36oto 46oF). Avoid shaking.
9.3.4 Administration - Subcutaneous injection is preferred. If clinically indicated, filgrastim may be administered as an intravenous infusion over 15 to 30 minutes.  
9.3.5 Toxicities - Medullary bone or skeletal pain is the most commonly reported toxicity. In addition, reversible elevations in uric acid, lactate dehydrogenase, and alkaline phosphatase are common laboratory abnormalities. Four cases of splenic rupture have been reported in healthy donors when given filgrastim or other myeloid growth factors for peripheral blood stem cell mobilization; 1 of these cases resulted in fatality. Five additional cases of splenic rupture have been reported in cancer patients undergoing chemotherapy or peripheral blood stem cell mobilization; splenic rupture may have contributed to deaths in 2 of these cases. One additional death due to splenic rupture after filgrastim therapy was reported to the manufacturer without additional information. According to the manufacturer, the reporting rate for splenic rupture with filgrastim is less than 1 in 486,000.  
9.4 CYCLOSPORINE (GENGRAF, SANDIMMUNE, NEORAL) 9.4.1 Supply - Cyclosporine will be obtained by the NIH Clinical Center Pharmacy Department from commercial sources and is available in capsules (25 mg and 100 mg), USP [MODIFIED], oral solution (100 mg/ml), USP [MODIFIED], and as a parenteral concentrate for injection (50 mg/ml). When oral capsules are prescribed for this protocol, the cyclosporine capsules, USP [NON-MODIFIED] should NOT be used.  
9.4.2 Preparation - For parenteral doses, each milliliter of concentrate (50mg/ml) should be diluted in 20 to 100ml of dextrose 5% in water or sodium chloride 0.9%. Parenteral doses of cyclosporine will be prepared in non-PVC containers and infused with non-PVC administration sets/tubing (see Storage and Stability, section 8.4.3). Oral cyclosporine solution may be mixed in orange juice or other beverages, but not milk.  
9.4.3 Storage and Stability - Capsules, oral solution, and ampules of parenteral concentrate bear expiration dates and are stored at room temperature and protected from light. Cyclosporine concentrate for injection that has been diluted to a final concentration of approximately 2mg/ml is stable for 24 hours in 5% dextrose or 0.9% sodium chloride injection in glass, PVC or non-PVC plastic containers. To minimize the potential for sorption to PVC plastic bags and tubing as well the leaching of phthalate plasticizer (DEHP) into the solution, only non-PVC plastic bags and intravenous administration sets should be utilized.  
9.4.4 Administration - Cyclosporine may be given intravenously over 2 hours or orally. 9.4.5 Toxicities - Acute cyclosporine nephrotoxicity is usually manifested by a moderate decline in renal excretory function, which is readily reversible by a decrease in drug dosage. Although some degree of transient renal dysfunction may occur in patients with therapeutic levels of cyclosporine, significant renal toxicity is associated with elevated trough levels. In addition to an increase in BUN and creatinine, hyperkalemic hyperchloremic acidosis, low fractional excretion of sodium and the onset of hypertension with hypomagnesemia are seen with cyclosporine nephrotoxicity. Hypertension occurs in up to 60% of patients. Hypomagnesemia can be associated with neurologic symptoms, including seizures, cerebellar ataxia and depression. Dose-related hepatotoxicity, manifested by elevation of serum transaminases and bilirubin, has been reported.
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9.5 fludarabine (fludara berlex laboratories)

9.5.1 Supply - Fludarabine monophosphate will be purchased by the NIH Clinical Center Pharmacy Department from commercial sources and is supplied as a white, lyophilized powder. Each vial contains 50 mg of fludarabine phosphate, 50 mg of mannitol, and sodium hydroxide to adjust pH. Fludara is stored at room temperature.  
9.5.2 Preparation - FLUDARA IV should be prepared for parenteral use by aseptically adding Sterile Water for Injection, USP. When reconstituted with 2 ml of Sterile Water for Injection, each ml of the resulting solution will contain 25 mg of Fludarabine Phosphate, 25 mg of mannitol, and sodium hydroxide to adjust the pH to 7–8.5. Fludarabine will be diluted in 100 to 125ml of either 5% dextrose in water or 0.9% sodium chloride, and infused IV over 30 minutes.  
9.5.3 Storage and Stability - Reconstituted FLUDARA IV is chemically and physically stable for 24 hours at room temperature or for 48 hours if refrigerated. Because reconstituted FLUDARA IV contains no antimicrobial preservative, care must be taken to assure the sterility of the prepared solution; for this reason, reconstituted FLUDARA IV should be used or discarded within 8 hours.  
9.5.4 Administration - Parenteral drug products should be inspected visually for particulate matter and discoloration prior to administration.
9.5.5 Toxicities - Fludarabine toxicities include myelosuppression (dose limiting toxicity),
fever, nausea, vomiting, stomatitis, diarrhea, gastrointestinal bleeding, anorexia, edema, skin rashes, myalgia, headache, agitation, hearing loss, transient episodes of somnolence and fatigue, auto-immune hemolytic anemia, auto-immune thrombocytopenia, paresthesias, peripheral neuropathy, renal, and pulmonary toxicity (interstitial pneumonitis). Severe fatal CNS toxicity presenting with loss of vision and progressive deterioration of mental status were encountered almost exclusively after very high doses of fludarabine monophosphate. Such toxicity has only rarely been demonstrated at the 25-30 mg/m2/day dosage of fludarabine. Very rarely described complications include transfusion-associated graft-versus-host disease, thrombotic thrombocytopenic purpura, and liver failure. Tumor lysis syndrome following fludarabine administration has been observed, especially in patients with advanced bulky disease. Opportunistic infections (protozoan, viral, fungal, and bacterial) have been observed post-fludarabine, especially in heavily pre-treated individuals, and in individuals receiving fludarabine combined with other agents.  
9.6 ETOPOSIDE, DOXORUBICIN, AND VINCRISTINE (IN EPOCH) 9.6.1 Supply - Etoposide, doxorubicin, and vincristine will be purchased by the NIH Clinical Center Pharmacy Department from commercial sources.  
9.6.2 Preparation and Administration - Etoposide, doxorubicin, and vincristine will be combined in a single ('3-in-1') admixture [108]. All 3-in-1 admixtures dispensed from the Pharmacy will contain a 24-hour supply of etoposide, doxorubicin, and vincristine PLUS 40 mL overfill (excess) fluid and a proportional amount of drug to compensate for volume lost in administration set tubing.  
| Etoposide      | Volume of Fluid          |                     |                   |                      |
|----------------|--------------------------|---------------------|-------------------|----------------------|
| Dose           | Containing a  Daily Dose | Volume of  Overfill | Volume To  Infuse | Administration  Rate |
| (fluid + drug) | (over 24 hours)          |                     |                   |                      |
| < 130 mg       | 528 mL                   | 40 mL               | 528 mL            | 22 mL/hour           |
| ≥ 130 mg       | 1056 mL                  | 40 mL               | 1056 mL           | 44 mL/hour           |  
Before dispensing 3-in-1 admixtures, Pharmacy staff will (1) purge all air from the drug product container, (2) attach an administration set appropriate for use with a portable pump and the set will be (3) primed close to its distal tip and (4) capped with a Luerlocking cap. Bags will be exchanged daily for 4 consecutive days to complete a 96hour drug infusion. Portable pumps used to administer etoposide + doxorubicin + vincristine admixtures will be programmed to deliver one of two fixed volumes at one of two corresponding fixed rates based on the amount of etoposide and fluid that is ordered (see table, above). At the end of an infusion, some residual fluid is expected because overfill fluid and drug were added; however, nurses are asked to return to the Pharmacy for measurement any drug containers that appear to contain a greater amount of residual drug than expected.  
9.6.3 Storage and Stability - Studies conducted by the Pharmaceutical Development Service, Pharmacy Department, NIH Clinical Center, have demonstrated that admixtures of vincristine, doxorubicin, and etoposide in 0.9% Sodium Chloride for Injection (at concentrations, respectively, of 1, 25, and 125 mcg/ml; 2, 50, and 250 mcg/ml; and 2.8, 70, and 350 mcg/ml) are stable for at least 48 hours at room temperature when protected from light [108].  
9.6.4 Toxicities:
9.6.4.1 Etoposide: nausea, vomiting, stomatitis, diarrhea, thrombocytopenia, neutropenia, and alopecia. Secondary AML has been associated with
etoposide. Bradycardia and hypotension are sometimes observed with etoposide administration.  
9.6.4.2 Doxorubicin: Cardiotoxicity is particularly noted after cumulative doses of greater than 550 mg/m2. Other toxicities include myelosuppression, nausea, vomiting, stomatitis, diarrhea, and alopecia. Skin infiltration of doxorubicin causes tissue necrosis.  
9.6.4.3 Vincristine causes neurological toxicities such as paresthesias, jaw pain, ataxia, foot-drop, cranial nerve palsies, paralytic ileus, constipation, abdominal pain, and loss of deep tendon reflexes. It is also a vesicant, and occasionally causes alopecia and myelosuppression.
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9.7 prednisone

9.7.1 Supply - Prednisone will be purchased by the NIH Clinical Center Pharmacy Department from commercial sources. Prednisone is commercially available as tablets, in strengths of 1, 2.5, 5, 20, and 50 mg.  
9.7.2 Storage and Stability - Prednisone tablets should be stored in the container provided away from heat. The product labeling bears the manufacturer's expiration dating for stability.  
9.7.3 Administration - Prednisone will be administered at a dose of 60 mg/m2orally on days 1, 2, 3, 4, and 5 of the induction chemotherapy regimen. In patients unable to tolerate oral medication, methylprednisolone can be substituted at an equivalent dosage, diluted in a small volume (e.g. 25-50ml) of 5% dextrose in water or 0.9% sodium chloride and infused over 15 minutes. To reduce gastrointestinal side effects, prednisone should be taken with food.  
9.7.4 Toxicities - Prednisone frequently causes gastritis, immunosuppression, muscle wasting, fluid retention, and hyperglycemia.
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9.8 rituximab (rituxan)

9.8.1 Supply - Rituximab will be obtained commercially and is supplied as a 10 mg/ml sterile, preservative-free solution for injection, in vials of 100 mg and 500 mg.  
9.8.2 Preparation - The desired dose will be diluted to a final concentration of 1 to 4 mg/mL in either 0.9% Sodium Chloride or D5W.  
9.8.3 Storage and Stability - Rituximab vials should be stored at 2–8°C (36–46°F) and should be protected from direct sunlight.  
9.8.4 Administration - First Infusion: The rituximab solution for infusion should be administered intravenously at an initial rate of 50 mg/hr. Rituximab should not be mixed or diluted with other drugs. If hypersensitivity or infusion reactions do not occur, escalate the infusion rate in 50 mg/hr increments every 30 minutes, to a maximum of 400 mg/hr. If hypersensitivity (non-IgE-mediated) or an infusion reaction develops, the infusion should be temporarily slowed or interrupted. The infusion can continue at onehalf the previous rate upon improvement of patient symptoms. If the first infusion is tolerated well, infusions during subsequent cycles of EPOCH-F/R can begin at a rate of 100 mg/hr and be increased by 100 mg/hr increments at 30-minute intervals. However, if the first infusion is not tolerated well, then the guidelines for the initial infusion should be followed for subsequent administration. Pre-medication will routinely be administered 30 to 60 minutes prior to the beginning of each rituximab infusion, consisting of acetaminophen 650 mg PO and diphenhydramine 25 to 50 mg i.v./p.o..  
9.8.5 Toxicities - The most serious adverse reactions caused by rituximab include infusion reactions, tumor lysis syndrome, mucocutaneous reactions, hypersensitivity reactions, cardiac arrhythmias and angina, and renal failure.  
9.8.5.1 *Fatal and Severe Infusion Reaction*s: Deaths within 24 hours of rituximab infusion have been reported. Approximately 80% of fatal infusion reactions occurred in association with the first infusion. Severe reactions typically occurred during the first infusion with time to onset of 30 to 120 minutes. Signs and symptoms of severe infusion reactions may include hypotension, angioedema, hypoxia or bronchospasm, and may require interruption of rituximab administration. The most severe manifestations and sequelae include pulmonary infiltrates, acute respiratory distress syndrome, myocardial infarction, ventricular fibrillation, and cardiogenic shock. In the reported cases, the following factors were more frequently associated with fatal outcomes: female gender, pulmonary infiltrates, and chronic lymphocytic leukemia or mantle cell lymphoma.  
9.8.5.2 *Management of severe infusion reactions:* The rituximab infusion should be interrupted for severe reactions and supportive care measures instituted as medically indicated (e.g., intravenous fluids, vasopressors, oxygen, bronchodilators, diphenhydramine, and acetaminophen). In most cases, the infusion can be resumed at a 50% reduction in rate (e.g., from 100 mg/hr to 50 mg/hr) when symptoms have completely resolved. Patients requiring close monitoring during first and all subsequent infusions include those with preexisting cardiac and pulmonary conditions, those with prior clinically significant cardiopulmonary adverse events and those with high numbers of circulating malignant cells (= 25,000/mm3) with or without evidence of high tumor burden.  
9.8.5.3 *Tumor Lysis Syndrome (TLS)*: Acute renal failure requiring dialysis with instances of fatal outcome has been reported in the setting of TLS following treatment with rituximab. Rapid reduction in tumor volume followed by acute renal failure, hyperkalemia, hypocalcemia, hyperuricemia, or hyperphosphatasemia, have been reported within 12 to 24 hours after the first rituximab infusion. Rare instances of fatal outcome have been reported in the setting of TLS following treatment with rituximab. The risks of TLS appear to be greater in patients with high numbers of circulating malignant cells (=
25,000/mm3) or high tumor burden. Prophylaxis for TLS should be considered for patients at high risk. Correction of electrolyte abnormalities, monitoring of renal function and fluid balance, and administration of supportive care, including dialysis, should be initiated as indicated. Following complete resolution of the complications of TLS, rituximab has been tolerated when readministered in conjunction with prophylactic therapy for TLS in a limited number of cases.  
9.8.5.4 *Severe Mucocutaneous Reactions*: Mucocutaneous reactions, some with fatal outcome, have been reported in patients treated with rituximab. These reports include paraneoplastic pemphigus (an uncommon disorder which is a manifestation of the patient's underlying malignancy), Stevens-Johnson syndrome, lichenoid dermatitis, vesiculobullous dermatitis, and toxic epidermal necrolysis. The onset of the reaction in the reported cases has varied from 1 to 13 weeks following rituximab exposure. Patients experiencing a severe mucocutaneous reaction should not receive any further infusions and seek prompt medical evaluation. Skin biopsy may help to distinguish among different mucocutaneous reactions and guide subsequent treatment. The safety of re-administration of RITUXAN to patients with any of these mucocutaneous reactions has not been determined.  
9.8.5.5 Infusion reactions and lymphopenia are the most commonly occurring adverse reactions. Mild to moderate infusion reactions consisting of fever and chills/rigors occur in the majority of patients during the first rituximab infusion. Other frequent infusion reaction symptoms included nausea, pruritus, angioedema, asthenia, hypotension, headache, bronchospasm, throat irritation, rhinitis, urticaria, rash, vomiting, myalgia, dizziness, and hypertension. These reactions generally occur within 30 to 120 minutes of beginning the first infusion, and resolved with slowing or interruption of the rituximab infusion and with supportive care (diphenhydramine, acetaminophen, IV saline, and vasopressors). In an analysis of data from 356 patients with relapsed or refractory, low-grade NHL who received 4 (N = 319) or 8 (N = 37) weekly infusions of rituximab, the incidence of infusion reactions was highest during the first infusion (77%) and decreased with each subsequent infusion (30% with fourth infusion and 14% with eighth infusion).
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9.9 cytarabine (cytosine arabinoside cytosar

9.9.1 Supply - Cytarabine will be purchased by the NIH Clinical Center Pharmacy Department from commercial sources and is supplied as un-reconstituted lyophilized powder (100 mg, 500 mg, 1 g, and 2 g vials). Cytarabine for injection is available for subcutaneous, intravenous, and intrathecal administration.  
9.9.2 Preparation - To prepare high dose (2 gm/m2) cytarabine intravenous infusions, each vial of drug should be reconstituted with sterile water for injection USP (containing no preservative) with the diluent volumes listed below. Note that the manufacturers recommended diluent for cytarabine is bacteriostatic water for injection containing benzyl alcohol. For preparation of intrathecal injection doses and for high dose intravenous dosing regimens, ONLY a preservative-free diluent should be used. The recommended volumes for reconstitution include the following:  
| Vial Size   | Volume of Diluent   | Concentration   |
|-------------|---------------------|-----------------|
| 100 mg      | 5 ml                | 20 mg/ml        |
| 500 mg      | 10 ml               | 50 mg/ml        |  
2 g 20 ml 100 mg/ml The drug will be further diluted in 500 ml of 5% Dextrose Injection or 0.9% Sodium Chloride Injection prior to administration.  
9.9.3 Storage and Stability - Vials should be stored at room temperature (25oC or 77oF);
excursions permitted to 15-30o C (59-86oF). [See USP Controlled Room Temperature.]
Once reconstituted with sterile water for injection, USP the reconstituted vial should be used within 24 hours. After final dilution with 0.9% Sodium Chloride Injection or 5% Dextrose Injection, the prepared dose should be used within 24 hours if stored at room temperature or 48 hours if refrigerated.  
9.9.4 Administration - Begin each cytarabine dose 3.5 hours after completion of the preceding fludarabine dose. Intravenous doses should be infused over at least 2 hours.  
9.9.5 Toxicity - Acute dose-limiting toxicity with IV administration consists of severe leukopenia and thrombocytopenia. Nausea and vomiting may be dose limiting at higher doses. Other adverse reactions include diarrhea, immunosuppression, anorexia, stomatitis, oral ulceration, flu-like syndrome, fever, hepatic dysfunction, and alopecia. At high doses, as in this protocol, keratoconjunctivitis, dermatitis, and central nervous system toxicity (e.g., ataxia, somnolence, coma, dysarthria) may occur. Occasionally, the CNS impairment is not fully reversible. Renal impairment will enhance toxicity. In the event that signs of CNS toxicity occur, the cytarabine will be interrupted and the M.D. notified. No further cytarabine will be administered if there is CNS toxicity (any grade) deemed related to cytarabine.  
9.9.6 Conjunctivitis Prophylaxis: Corticosteroid ophthalmic drops will be administered 2 drops to each eye every 6 hours starting prior to first dose and continuing until 24 hours after the last dose of cytarabine has been completed.
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9.10 diphenhydramine

9.10.1 Supply - Commercially available. Diphenhydramine HCl injection is available in an injectable solution at a 50mg/ml concentration in single dose ampules, syringes and vials as well as multi-dose vials from multiple manufacturers.  
9.10.2 Preparation - Diphenhydramine HCl may be given by direct intravenous injection without additional dilution. Alternatively the prescribed dose may be diluted in a small volume (e.g. 25-50ml) of 5% dextrose in water (D5W) or 0.9% sodium chloride (NS) and infused over 10-15 minutes.  
9.10.3 Storage and Stability - Store commercially available injectable product at controlled room temperature.  
9.10.4 Administration - Diphenhydramine HCl injection may be administered by direct IV
injection (IV push) at a rate generally not exceeding 25mg/min. Alternatively, diphenhydramine HCl injection may be diluted and given over 10-15 minutes (see Preparation).  
9.10.5 Toxicities - Sedation, sleepiness, dizziness, disturbed coordination, epigastric distress, thickening of bronchial secretions. Diphenhydramine can provide additive effects with alcohol or other CNS depressants. Diphenhydramine can cause anticholinergic side effects (e.g. dry mouth, fixed or dilated pupils, flushing, urinary retention). Diphenhydramine should be used with caution in patients with a history of bronchial asthma, increased intraocular pressure, hyperthyroidism, cardiovascular disease or hypertension.
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9.11 acetaminophen

9.11.1 Supply - Commercially available as 325 mg or 500 mg tablets for oral administration from multiple manufacturers.  
9.11.2 Storage - Store at controlled room temperature. 9.11.3 Administration - Oral. For analgesia and antipyresis, the usual dose is 650 to 1000 milligrams every 4 to 6 hours, to a maximum of 4 grams/day.  
9.11.4 Toxicities - No toxicities are anticipated to result from single doses of acetaminophen administered as pre-medication for rituximab infusions.
9.12 VALACYCLOVIR (VALTREX®)  
9.12.1 Supply - Commercially available as 500mg tablets and 1gm tablets. Dose adjustment is necessary in patients with significant renal impairment (refer to the manufacturer's labeling for dose adjustment guidelines.)
9.12.2 Pharmacology - Valacyclovir is the hydrochloride salt of L-valyl ester of the antiviral drug acyclovir. After oral administration, valacyclovir is rapidly absorbed from the GI tract and nearly completely converted to acyclovir and L-valine by first-pass intestinal or hepatic metabolism.  
9.12.3 Storage and Stability - Oral tablets should be stored at 15oto 25oC (59oto 77oF).
9.12.4 Administration - Oral. 9.12.5 Toxicities - Nausea and/or vomiting, headache, dizziness, abdominal pain, dysmenorrhea, arthralgia, acute hypersensitivity reactions, elevations in liver enzyme laboratory values (e.g. AST). Renal failure and CNS symptoms have been reported in patients with renal impairment who received valacyclovir or acyclovir at greater than the recommended dose. Dose reduction is recommended in this patient population (refer to the manufacturer's labeling for dose adjustment guidelines).
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9.13 fluconazole (diflucan)

9.13.1 Supply - Commercially available as 50 mg, 100 mg, 150 mg and 200 mg tablets, or as a powder for oral suspension for reconstitution at a concentration of 10mg/ml or 40 mg/ml. Parenteral fluconazole is available in a solution for injection at a concentration of 2 mg/ml in glass bottles and Viaflex® Plus plastic containers containing either 100ml (200 mg) or 200ml (400 mg).  
9.13.2 Preparation - For parenteral administration, the commercial solution for injection is available in its final form for administration (concentration of 2 mg/ml).  
9.13.3 Storage and Stability - Oral tablets and oral suspension should be stored at temperatures below 30oC (86oF). Store reconstituted oral suspension between 5oto 30oC (41oto 86oF)
and discard unused portion after 2 weeks. Fluconazole for injection in glass bottles should be stored between 5oto 30oC (41oto 86oF). Fluconazole for injection in Viaflex® Plus plastic containers should be stored between 5oto 25oC (41oto 77oF).  
9.13.4 Administration - Oral and parenteral. Parenteral doses should be administered by an intravenous infusion at a maximum rate of 200mg/hr.  
9.13.5 Toxicities - Nausea, vomiting, headache, skin rash, abdominal pain, diarrhea have been reported at an incidence of 1% or greater in clinical trials. In combined clinical trials and marketing experience, there have been rare cases of serious hepatic reactions during treatment with fluconazole. The spectrum of these hepatic reactions has ranged from mild transient elevation in transaminases to clinical hepatitis, cholestasis and fulminant hepatic failure, including fatalities.  
9.13.6 Drug Interactions - Fluconazole is a potent inhibitor of the cytochrome P450 3A4 isoenzyme system. Co-administration of fluconazole with other drugs metabolized by the same enzyme system may result in increased plasma concentrations of the drugs, which could increase or prolong therapeutic and adverse effects. Refer to the package literature or other drug information resources for additional information on identification and management of potential drug interactions.  
9.14 TRIMETHOPRIM/SULFAMETHOXAZOLE (TMP/SMX, COTRIMOXAZOLE, BACTRIM,
SEPTRA)
9.14.1 Supply - Commercially available as a single strength tablet containing trimethoprim 80mg and sulfamethoxazole 400mg and a double strength (DS) tablet containing trimethoprim 160mg and sulfamethoxaole 800mg. It is also available in a oral suspension at a concentration of 40mg of trimethoprim and 200mg sulfamethoxazole per 5ml. Parenteral TMP/SMX is available in a solution for injection at a concentration of 80mg of trimethoprim and 400mg of sulfamethoxazole per 5ml.  
9.14.2 Preparation - For parenteral administration, the commercial solution for injection must be diluted prior to administration. It is recommended that each 5ml of the solution for injection be diluted with 100-125 ml or, if fluid restriction is required, in 75ml of dextrose 5% in water. 0.9% sodium chloride, Inj. may be substituted as a diluent but the resulting solutions have reduced stability. Consult with pharmacy for questions regarding diluent, volume, and expiration.  
9.14.3 Storage and Stability - Oral tablets and oral suspension should be stored at 15oto 30oC
(59oto 86oF) in a dry place and protected from light. TMP/SMX for injection should be stored at room temperature between 15oto 30oC (59oto 86oF) and should not be refrigerated. Stability of intravenous doses after final dilution is dependent on concentration and diluent. Consult with pharmacy for questions regarding stability and expiration dating.  
9.14.4 Administration - Oral and parenteral. Parenteral doses should be administered by an intravenous infusion over 60 to 90 minutes.  
9.14.5 Toxicities - The most common adverse effects from TMP/SMX are gastrointestinal disturbances (nausea, vomiting, anorexia) and allergic skin reactions (such as rash and urticaria). Fatalities associated with the administration of sulfonamides, although rare, have occurred due to severe reactions, including Stevens-Johnson syndrome, toxic epidermal necrolysis, fulminant hepatic necrosis, agranulocytosis, aplastic anemia and other blood dyscrasias. For TMP/SMX injection, local reaction, pain and slight irritation on IV administration are infrequent. Thrombophlebitis has rarely been observed.
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9.15 furosemide (lasix)

9.15.1 Supply - Commercially available as 20mg, 40mg, and 80mg tablets or as an injectable solution of 10mg/ml, from multiple manufacturers.  
9.15.2 Preparation - Furosemide may be given by direct intravenous injection without additional dilution. Alternatively the prescribed dose may be diluted in a small volume (e.g. 2550ml) of 5% dextrose in water (D5W) or 0.9% sodium chloride (NS).  
9.15.3 Storage and Stability - Store commercially available injectable product at controlled room temperature and protect from light exposure.  
9.15.4 Administration - Furosemide injection may be administered by direct IV injection (IV
push) over 1 to 2 minutes, and by intravenous infusion at a rate not exceeding 4mg/min.
9.15.5 Toxicities - Common toxicities include hypokalemia, hypomagnesemia, hyperuricemia, hyperglycemia, glycosuria, anorexia, constipation, cramping, diarrhea, blurred vision, dizziness, headache, parathesias, vertigo, muscle spasms, weakness, purpura, photosensitivity, pruritus, urticaria, and rash. Uncommon but serious adverse effects include cytopenias, hemolytic anemia, erythema multiforme, Stevens Johnson syndrome, hypotension, and pancreatitis.  
9.16 METHOTREXATE (METHOTREXATE SODIUM, MTX, NSC-740) 9.16.1 Supply - Methotrexate will be obtained commercially and is supplied as a 25 mg/ml preservative-free isotonic solution for injection.  
9.16.2 Preparation - The desired dose will be diluted in 5% dextrose in water or 0.9% sodium chloride.
9.16.3 Storage and Stability - Methotrexate should be stored at room temperature and protected from light. Once the prepared dose is diluted for administration, the solution is stable for 24 hours refrigerated or at room temperature when protected from light.  
9.16.4 Administration - Methotrexate will be given as IV infusion over 15 minutes. 9.16.5 Toxicities - The toxicity associated with methotrexate primarily involves the gastrointestinal tract. Severe mucositis can occur, particularly in patients who have received an intensive preparative regimen. Other gastrointestinal symptoms include nausea, vomiting and diarrhea. Transient elevations in serum transaminases have been seen. The myelosuppression associated with methotrexate often results in delayed engraftment. Folinic acid can effectively circumvent the enzymatic block produced by methotrexate.  
9.17 SIROLIMUS (RAPAMYCIN) (RAPAMUNE®, WYETH-AYERST LABORATORIES) 9.17.1 Supply - For patient administration, oral tablets will be purchased by the NIH Clinical Center Pharmacy Department from commercial sources.  
9.17.2 Storage and Stability - Oral tablets should be stored 20-25 C° (68-77 F°). Oral solution should be refrigerated (2-8 C° or 36-46 F°).
9.17.3 Administration: Because food, in particular fatty foods, can decrease the absorption of sirolimus, it is suggested the tablets should be administered consistently between meals.  
9.17.4 Toxicities: 1) Sirolimus induces immune suppression, which has been associated with opportunistic infection and an increased rate of malignancy, particularly skin cancer. 2) Some individuals may develop hypersensitivity to sirolimus. 3) May cause an increase in cholesterol and triglycerides, which may be associated with pancreatitis. 4) With longterm administration, may result in impaired renal function. 4) Metabolism is via the cytochrome p450 pathway, and as such, co-administration of voriconazole is prohibited with sirolimus can result in prolonged blood levels of sirolimus. As such, if the administration of voriconazole is required the dose of sirolimus will be reduced by 90%
and close attention must be given relative to serum sirolimus levels and associated toxicities [98].
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9.18 alemtuzumab (campath-1h campath)

9.18.1 Supply –Available through the Campath Distribution Program (The Sanofi Foundation for North America 1-877-422-6728). Vials are provided through this program upon completion of a patient specific request form. Prior to submission of a drug request the patient must provide authorization for the release of medical information (NIH-527). Refer to the Pharmacy Department or Clinical Pharmacy Specialist for additional details on drug procurement. Each single use, clear glass vial of Campath contains 30 mg Alemtuzumab in 1 mL of solution (8.0 mg sodium chloride, 1.44 mg dibasic sodium phosphate, 0.2 mg potassium chloride, 0.2 mg monobasic potassium phosphate, 0.1 mg polysorbate 80, and .0187 mg disodium edetate dehydrate). No preservatives are added. Each carton contains three Campath vials (NDC 50419-357-03) or one Campath vial (NDC 50419-357-01).  
9.18.2 Storage and stability - Vials of Alemtuzumab should be stored at 2-8°C (36-46°F) and protected from sunlight. The vial should not be frozen; if the vial has been frozen it should be discarded. Alemtuzumab contains no antimicrobial preservative. An internal NIH Pharmacy (Pharmaceutical Development Section) conducted study demonstrated 24 hour stability of alemtuzumab when diluted in 0.9% sodium chloride to a concentration range of 6.67 mcg/mL to 120 mcg/mL at room temperature (Goldspiel JT, et.al. Stability of alemtuzumab solutions at room temperature. Am J Health –Syst Pharm, accepted for publication 2012) .. Alemtuzumab solutions prepared in the concentration range described above may be stored at room temperature (15-30°C) for up to 24 hours. Alemtuzumab solutions should be protected from light.  
9.18.3 Preparation - The vial should be inspected for visible particulate matter and discoloration prior to administration. If particulate matter is present or the solution is discolored, the vial should not be used. The vial should not be shaken prior to use. The necessary amount of Alemtuzumab should be withdrawn from the vial into a syringe. The vial contains no preservatives and is intended for single use only; the vial should be discarded with any unused portion after 6 hours. The desired dose of 20 mg is then injected into 250 mL sterile 0.9% Sodium Chloride USP. The bag should be gently inverted to mix the solution. The syringe is discarded.  
9.18.4 Administration - Alemtuzumab should be administered intravenously only. The infusion should be administered over an 8 hour period.. Other drug substances should not be added or simultaneously infused through the same intravenous line.  
9.18.5 Toxicities:
9.18.5.1 Infusion-related adverse events are very common (>80%) with Alemtuzumab administration. The most commonly reported infusion-related adverse events on this study included rigors, fever, nausea, vomiting, and hypotension. Other frequently reported infusion-related events include rash, fatigue, urticaria, dyspnea, pruritus, headache, and diarrhea. Serious infusion-related events that have been reported include syncope, pulmonary infiltrates, ARDS, respiratory arrest, cardiac arrhythmias, myocardial infarction and cardiac arrest.  
9.18.5.2 The following serious adverse events have been were reported in at least one patient treated on studies where Alemtuzumab was used as a single agent to treat malignant (e.g. chronic lymphocytic leukemia) and non-malignant diseases (e.g. rheumatoid arthritis): allergic and anaphylactoid reactions, ascites, cyanosis, atrial and ventricular arrhythmias, angina pericarditis, abnormal gait, aphasia, coma, seizures, hyperthyroidism, duodenal ulcer, GI hemorrhage, intestinal obstruction, intestinal perforation, pancreatitis, hepatocellular damage, respiratory alkalosis, arthritis or worsening arthritis, arthropathy, osteomyelitis, polymyositis, malignant lymphoma, secondary leukemia, disseminated intravascular coagulation, thrombocythemia agranulocytosis, aplasia, lymphadenopathy, hemolytic anemia, splenic infarction, splenomegaly, cervical dysplasia, bacterial infections, *Herpes zoster* infection, *Pneumocystis carinii* infection, otitis media, asthma, pleural effusion, pleurisy, pulmonary edema, pulmonary fibrosis, acute renal failure, cerebral hemorrhage, and deep vein thrombosis.  
9.19 URSODEOXYCHOLIC ACID (URSODIOL, ACTIGALL®)
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9.19.1 supply - commercially available

9.19.2 Pharmacology - About 90% of a therapeutic dose of Ursodiol is absorbed in the small bowel after oral administration. After absorption, Ursodiol enters the portal vein and undergoes efficient extraction from portal blood by the liver where it is conjugated with either glycine or taurine and is then secreted into the hepatic bile ducts. Only small quantities of Ursodiol appear in the systemic circulation and very small amounts are excreted into urine. The sites of the drug's therapeutic actions are in the liver, bile, and gut lumen.  
9.19.3 Storage and Stability - Oral capsules should be stored at 15°C-30°C (59°F-86°F).  
9.19.4 Administration - Oral. 9.19.5 Toxicities - Nausea, vomiting, dyspepsia, metallic taste, abdominal pain, biliary pain, cholecystitis, constipation, stomatitis, flatulence, diarrhea, pruritus, rash, dry skin,
urticaria, headache, fatigue, anxiety, depression, sleep disorders. Less common side effects include sweating, thinning of hair, back pain, arthralgia, myalgia, rhinitis, cough.  
9.19.6 Drug Interactions - Bile acid sequestering agents such as cholestyramine and colestipol may interfere with the action of Ursodiol by reducing its absorption. Aluminum-based antacids have been shown to adsorb bile acids in vitro and may be expected to interfere with Ursodiol in the same manner as the bile acid sequestering agents. Estrogens, oral contraceptives, and clofibrate increase hepatic cholesterol secretion, and encourage cholesterol gallstone formation and hence may counteract the effectiveness of Ursodiol.  
9.20 TACROLIMUS (FK-506, PROGRAF®) 9.20.1 Supply - Tacrolimus will be obtained by the NIH Clinical Center Pharmacy Department from commercial sources and is available for oral administration as capsules (tacrolimus capsules) containing the equivalent of 0.5 mg, 1 mg or 5 mg of anhydrous tacrolimus. Tacrolimus is also available as a sterile solution (tacrolimus injection) containing the equivalent of 5 mg anhydrous tacrolimus in 1 mL for administration by intravenous infusion only. Each mL contains polyoxyl 60 hydrogenated castor oil (HCO-60), 200 mg, and dehydrated alcohol, USP, 80.0% v/v.  
9.20.2 Preparation - For parenteral doses, tacrolimus injection must be diluted with 0.9%
Sodium Chloride Injection or 5% Dextrose Injection before use. Parenteral doses of tacrolimus will be prepared in non-PVC containers and infused with non-PVC administration sets/tubing (see Storage and Stability, section 8.4.3).  
9.20.3 Storage and Stability - Capsules, oral solution, and ampules of parenteral concentrate bear expiration dates and are stored at room temperature and protected from light.  
9.20.4 Administration - Tacrolimus will be administered as a continuous intravenous infusion over 24 hours or given orally in divided doses every 12 hours.
9.20.5 Toxicities - Tacrolimus can cause neurotoxicity and nephrotoxicity, particularly when used in high doses. Mild to severe hyperkalemia has been observed following tacrolimus administration. Serum potassium levels should be monitored and potassium-sparing diuretics should not be used during tacrolimus therapy. Neurotoxicity, including tremor, headache, and other changes in motor function,0 mental status, and sensory function have been reported with tacrolimus administration. Tremor and headache have been associated with high whole-blood concentrations of tacrolimus and may respond to dosage adjustment. Seizures have occurred in adult and pediatric patients receiving tacrolimus. Coma and delirium also have been associated with high plasma concentrations of tacrolimus. The most common adverse reactions reported with tacrolimus administration include infection, tremor, hypertension, abnormal renal function, constipation, diarrhea, headache, abdominal pain and insomnia. Less common adverse reactions reported with tacrolimus administration include anorexia, cholangitis, jaundice, diarrhea, dyspepsia, hepatitis, ALT (SGPT) increased, AST (SGOT) increased, abnormal ECG, angina pectoris, arrhythmia, acute kidney failure, acidosis, alkaline phosphatase increased, alkalosis, bicarbonate decreased, bilirubinemia, BUN increased, dehydration, edema, hypercalcemia, , hypocalcemia, hypercholesterolemia, hyperlipemia, hyperphosphatemia, hyperuricemiahypoglycemia, hypomagnesemia, hyponatremia, hypophosphatemia, hypoproteinemia, lactic dehydrogenase increase, peripheral edema, weight gain, Cushing's syndrome, diabetes mellitus, anemia, allergic reaction, and photosensitivity reaction.  
95
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Allogeneic stem cell transplantation after a fludarabine/busulfan-based reduced-intensity conditioning in patients with myelodysplastic syndrome or secondary acute myeloid leukemia. Ann Hematol. 2003; 82:336-42.  
89. Kobayashi K, Kami M, Murashige N, et al; Tokyo SCT Consortium Institution. Outcomes of patients with acute leukaemia who relapsed after reduced-intensity stem cell transplantation from HLA-identical or one antigen-mismatched related donors. Br J Haematol. 2005; 129:795-802.  
90. Hamaki T, Kami M, Kanda Y, et al. Reduced-intensity stem-cell transplantation for adult acute lymphoblastic leukemia: a retrospective study of 33 patients. Bone Marrow Transplant. 2005; 35:549-56.  
91. Schmid C, Schleuning M, Ledderose G, Tischer J, Kolb HJ. Sequential regimen of chemotherapy, reduced-intensity conditioning for allogeneic stem-cell transplantation, and prophylactic donor lymphocyte transfusion in high-risk acute myeloid leukemia and myelodysplastic syndrome. J Clin Oncol. 2005; 23:5675-87.  
92. Rondelli D, Barosi G, Bacigalupo A, et al; Myeloproliferative Diseases-Research Consortium. Allogeneic hematopoietic stem-cell transplantation with reduced-intensity conditioning in intermediate- or high-risk patients with myelofibrosis with myeloid metaplasia. Blood. 2005; 105:4115-9.  
93. Scott B, Deeg HJ. Hemopoietic cell transplantation as curative therapy of myelodysplastic syndromes and myeloproliferative disorders. Best Pract Res Clin Haematol. 2006; 19:51933.  
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Outcomes of reduced-intensity transplantation for chronic myeloid leukemia: an analysis of prognostic factors from the Chronic Leukemia Working Party of the EBMT. Blood. 2005; 106:2969-76.  
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Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood. 2002; 100:4325-36.  
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(IWG-MRT). International Working Group (IWG) consensus criteria for treatment response in myelofibrosis with myeloid metaplasia, for the IWG for Myelofibrosis Research and Treatment (IWG-MRT). Blood. 2006; 108:1497-503.  
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(TID) and Disease Control in Patients with Lymphoid Malignancies Prior To ReducedIntensity Allogeneic Hematopoietic Stem Cell Transplantation (RIST). Blood. 2006; 108:98a (Abstract \#317).  
101. Jackson G, Taylor P, Smith GM, et al. A multicentre, open, non-comparative phase II study of a combination of fludarabine phosphate, cytarabine and granulocyte colony-stimulating factor in relapsed and refractory acute myeloid leukaemia and de novo refractory anaemia with excess of blasts in transformation. Br J Haematol. 2001; 112:127-37.  
102. Marty FM, Lowry CM, Cutler CS, et al. Voriconazole and sirolimus coadministration after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant. 2006; 12:552-9.  
103. Essell JH, Schroeder MT, Harman GS, Halvorson R, Lew V, Callander N, Snyder M,
Lewis SK, Allerton JP, Thompson JM. Ursodiol prophylaxis against hepatic complications of allogeneic bone marrow transplantation. A randomized, double-blind, placebocontrolled trial. Ann Intern Med. 1998; 128:975-81.  
104. Ruutu T, Eriksson B, Remes K, Juvonen E, Volin L, Remberger M, Parkkali T, Hagglund H, Ringden O; Nordic Bone Marrow Transplantation Group. Ursodeoxycholic acid for the prevention of hepatic complications in allogeneic stem cell transplantation. Blood. 2002. 100(6):1977-83.  
105. Spitzer TR. Engraftment syndrome following hematopoietic stem cell transplantation.  
Bone Marrow Transplantation, 2001; 27:893-8.  
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Grading. Bone Marrow Transplant. 1995; 15:825-8.  
107. Bowden RA, Slichter SJ, Sayers M, Weisdorf D, Cays M, Schoch G, Banaji M, Haake R,
Welk K, Fisher L, McCullough J, Miller W.A comparison of filtered leukocyte-reduced and cytomegalovirus (CMV) seronegative blood products for the prevention of transfusionassociated CMV infection after marrow transplant. Blood. 1995; 86:3598-603.  
108. Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister TA, Vose J,
Grillo-Lopez A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos GP. Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group. J Clin Oncol. 1999; 17:1244. [erratum appears in J Clin Oncol 2000; 18:2351.].  
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Fisher RI, Hagenbeek A, Zucca E, Rosen ST, Stroobants S, Lister TA, Hoppe RT, Dreyling M, Tobinai K, Vose JM, Connors JM, Federico M, Diehl V; The International Harmonization Project on Lymphoma. Revised response criteria for malignant lymphoma. J Clin Oncol. 2007; 25:579-86.  
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112. Wolfe JL, et al. Compatibility and stability of vincristine sulfate, doxorubicin hydrochloride, and etoposide in 0.9% sodium chloride injection. Am J Health-System Pharm. 1999; 56:985-9.  
113. Empiric antifungal therapy in febrile granulocytopenic patients. EORTC International Antimicrobial Therapy Cooperative Group. Am J Med. 1989; 86:668-72.  
114. Walsh TJ, Pappas P, Winston DJ, et al; National Institute of Allergy and Infectious Diseases Mycoses Study Group. Voriconazole compared with liposomal amphotericin B for empirical antifungal therapy in patients with neutropenia and persistent fever. N Engl J Med. 2002; 346:225-34.  
115. Kanda Y, Mineishi S, Saito T, et al., Long-term low-dose acyclovir against varicella-zoster virus reactivation after allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant. 2001; 28:689-92.  
116. Steer CB, Szer J, Sasadeusz J, Matthews JP, Beresford JA, Grigg A. Varicella-zoster infection after allogeneic bone marrow transplantation: incidence, risk factors and prevention with low-dose aciclovir and ganciclovir. Bone Marrow Transplant. 2000; 25:657-64.  
117. Koc Y, Miller KB, Schenkein DP, Griffith J, Akhtar M, DesJardin J, Snydman DR.Varicella zoster virus infections following allogeneic bone marrow transplantation: frequency, risk factors, and clinical outcome. Biol Blood Marrow Transplant. 2000; 6:44-9.  
118. Nichols WG, Corey L, Gooley T, Drew WL, Miner R, Huang M, Davis C, Boeckh M.  
Rising pp65 antigenemia during preemptive anticytomegalovirus therapy after allogeneic hematopoietic stem cell transplantation: risk factors, correlation with DNA load, and outcomes. Blood. 2001; 97867-74.  
119. Kulkarni S, Powles R, Treleaven J, Riley U, Singhal S, Horton C, Sirohi B, Bhagwati N,
Meller S, Saso R, Mehta J. Chronic graft versus host disease is associated with long-term risk for pneumococcal infections in recipients of bone marrow transplants. Blood. 2000; 95:3683-6.  
120. Picardi M, Selleri C, Rotoli B. Spleen sizing by ultrasound scan and risk of pneumococcal infection in patients with chronic GVHD: preliminary observations. Bone Marrow Transplant. 1999; 24:173-7.  
121. Metcalf JP, Rennard SI, Reed EC, Haire WD, Sisson JH, Walter T, Robbins RA.  
Corticosteroids as adjunctive therapy for diffuse alveolar hemorrhage associated with bone marrow transplantation. University of Nebraska Medical Center Bone Marrow Transplant Group. Am J Med. 1994; 96:327-34.  
122. de Labarthe A, Rousselot P, Huguet-Rigal F, Delabesse E, Witz F, Maury S, Réa D,
Cayuela JM, Vekemans MC, Reman O, Buzyn A, Pigneux A, Escoffre M, Chalandon Y, MacIntyre E, Lhéritier V, Vernant JP, Thomas X, Ifrah N, Dombret H; Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL). Imatinib combined with induction or consolidation chemotherapy in patients with de novo Philadelphia chromosome-positive acute lymphoblastic leukemia: results of the GRAAPH-2003 study. Blood 109:1408-13, 2007 123. Oehler VG, Gooley T, Snyder DS, Johnston L, Lin A, Cummings CC, Chu S, Bhatia R,
Forman SJ, Negrin RS, Appelbaum FR, Radich JP. The effects of imatinib mesylate treatment before allogeneic transplantation for chronic myeloid leukemia. Blood 109:17829, 2007 124. Carpenter PA, Snyder DS, Flowers ME, Sanders JE, Gooley TA, Martin PJ, Appelbaum FR, Radich JP. Prophylactic administration of imatinib after hematopoietic cell transplantation for high-risk Philadelphia chromosome-positive leukemia. Blood 109:27913, 2007 125. Scholl S, Theuer C, Scheble, V, Kunert, C, Heller A, Mugge LO, Fricke HJ, Hoffken, K,
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Bornhauser M, Ritter M, Neubauer A, Ehninger G, Illmer T. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia; association with FAB subtypes and identification of subgroups with poor prognosis. Blood 2002; 99: 4326-35 127. Bornhauser M, Illmer T, Schaich M, Soucek S, Ehniger G, Thiede C. Improved outcome after stell-cell transplantion in FLT3/ITD-positive AML. Blood 2007; 109: 2264-5 128. Turgut B, Vural O, Pala FS, Pamuk GE, Tabakcioglu K, Demir M, Ongoren S, Soysal T,
Algunes C. 17p Deletion is associated with resistance of B-cell chronic lymphocytic leukemia cells to in vitro fludarabine-induced apoptosis. Leukemia and Lymphoma, 2007; 48 (2): 311-320  
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11 appendices

11.1 APPENDIX A - PROPHYLAXIS AND TREATMENT OF INFECTIOUS COMPLICATIONS IN
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allogeneic hsct recipients

(Note: The practice guidelines primarily follow the Guidelines for Infection Management as outlined by the NIH Blood and Marrow Transplant Consortium's Supportive Care Guidelines for Allogeneic Hematopoietic Stem Cell Transplant Recipients (http://intranet.cc.nih.gov/bmt/clinicalcare/guidelines.shtml). The practice guidelines included in this Appendix are based upon the best available clinical evidence and may change as additional data become available.) 11.1.1 Pneumocystis jiroveci pneumonia  
11.1.1.1 At the start of pre-transplant induction chemotherapy, all patients will receive trimethoprim 160 mg/sulfamethoxazole 800 mg (TMP/SMX 160/800), one tablet PO QD on three days per week. This will continue until completion of induction chemotherapy.  
11.1.1.2 Upon admission for the transplant conditioning regimen (day –7), all patients will receive TMP/SMX 160/800, one tablet PO BID, continuing daily through the evening of day –2.
11.1.1.3 Prophylaxis is resumed when the absolute neutrophil count is greater than 1,000/l and the platelet count is consistently above 50,000/mm3for two consecutive days without transfusion. Note: TMP/SMX should not be resumed before the last day of methotrexate administration (day +11 - see section 3.6.1.1.4). The dose for post-transplant prophylaxis is TMP/SMX 160/800, one tablet PO QD on three days per week, continuing until the recipient's CD4 count is above 200 cells/μl for 3 months and all immunosuppressive agents are discontinued.  
11.1.1.4 For patients allergic to sulfa, options include aerosolized pentamidine 300 mg by inhalation Q4 weeks, dapsone 100 mg PO QD, or atovaquone 1,500 mg PO QD. Prior to instituting dapsone therapy, G6PD deficiency must be excluded.  
11.1.2 FUNGAL PATHOGENS
11.1.2.1 Prophylaxis - Candida infections:
a) On day 6 of the first cycle of EPOCH-F/R induction chemotherapy, recipients will begin fluconazole 400 mg PO daily. Because of its interaction with vincristine, fluconazole will be discontinued during subsequent cycles of induction chemotherapy on days 1 through 5. Fluconazole will then be restarted on day 6 of each cycle.  
b) Recipients receiving FLAG induction chemotherapy will receive fluconazole 400 mg PO
daily, beginning with day 1 of the first cycle of FLAG.
c) Recipients will continue fluconazole 400 mg PO daily throughout the transplant conditioning regimen (day –6) until 100 days after transplantation and immunosuppression is discontinued. Intravenous dosing will be substituted when patients are unable to tolerate oral medications. Fluconazole will be discontinued when an alternative antifungal is started.  
d) Alternatives to fluconazole include caspofungin, posaconazole or itraconazole.  
Caspofungin dosing is 70 mg IV loading dose on day 1, then 50 mg IV QD. Posaconazole dosing is 200mg PO q 6-8h. Itraconazole dosing is 200 mg IV Q12 hours for 2 days (loading dose), then 200 mg IV Q24 hours for 12 days, then 200 mg PO Q12 hours to maintain levels (itraconazole plus hydroxy-itraconazole) greater than 1 μg/ml. Triazoles should be avoided in recipients with fluconazole hepatotoxicity. In such cases, consult the infectious diseases service.  
11.1.2.2 Prophylaxis - Aspergillus infections:
a) During Neutropenia: Neutropenic recipients who are febrile for more than 5 days despite broad-spectrum antibiotics and have no clinical evidence of invasive fungal disease will receive caspofungin 70 mg IV loading dose, then 50 mg IV Q24 hours. An alternative regimen is liposomal amphotericin B (Ambisome) 5 mg/kg/day or voriconazole 6 mg/kg q12h for two doses and then 4 mg/kg q12h. This therapy will continue until the patient attains an ANC > 500/μl AND is afebrile for 24 hours. Fluconazole prophylaxis will be discontinued during this therapy. Empiric antifungal therapy may be started at other times, if clinically indicated.  
b) During Steroid Therapy for GVHD: Recipients with GVHD receiving corticosteroids at a dose equivalent to > 1 mg/kg/day for > 6 days will receive posaconazole 200mg PO q68h. An alternative regimen is voriconazole 6 mg/kg IV Q12 hours x 2 doses, then 3 mg/kg IV Q12 hours. Voriconazole therapy will be converted to oral dosing at 200 mg PO Q12 hours when tolerated. Alternatively, voriconazole may be initiated with oral dosing at 400 mg PO Q12 hours x 2 doses, then 200 mg PO Q12 hours. Antifungal prophylaxis with activity against Aspergillus should be continued until the corticosteroid dose is reduced to < 0.6 mg/kg/day. Alternative regimens include:
- Amphotericin B lipid complex (ABLC) 5 mg/kg IV QD
- Caspofungin 70 mg IV loading dose followed by 50 mg IV QD - Itraconazole 200 mg IV Q12 hours x 2 days (loading dose), then 200 mg IV Q24 hours x 12 days. When tolerated, itraconazole may be converted to oral dosing at 200 mg PO Q12 hours to maintain levels (itraconazole + hydroxy-itraconazole) greater than 1 μg/ml.
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notes

Empirical antifungal therapy during neutropenia is controversial. Less than 10% of patients with persistent fever during neutropenia have a documented fungal infection. Only one RCT has shown decreased fungal infection-related death with the empirical addition of amphotericin B in neutropenic fever, and this study actually failed to show an overall survival advantage [113]. The relevance of these results to patients with neutropenia of short duration who are receiving fluconazole prophylaxis is questionable. In the most recent trial of empirical antifungal therapy, breakthrough fungal infections were more prevalent in high-risk patients with relapsed leukemia and/or allogeneic bone marrow transplant than in other febrile neutropenic patients [114]. If persistent fever during neutropenia is a manifestation of occult fungal infection in our patients (severely immunosuppressed, with short neutropenia and long-term use of fluconazole), then the most likely pathogens (fluconazole-resistant Candida and Aspergillus) are probably better treated with caspofungin or high-dose amphotericin B (at least 5 mg/kg). As our allogeneic HSCT recipients receive cyclosporine, a known nephrotoxic drug,after transplantation, amphotericin B lipid formulations are preferred. Voriconazole is a valid alternative, and in the mentioned study was associated with significantly fewer breakthrough fungal infections than Ambisome [114].
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11.1.3 viral pathogens

11.1.3.1 Herpes Simplex Virus (HSV) and Varicella Zoster Virus (VZV)
a) All patients will receive valacyclovir 500 mg PO QD for suppression of HSV and VZV
infection/reactivation. This prophylaxis will begin when recipients start induction chemotherapy (EPOCH-F/R) and continue throughout transplantation. Valacylovir will continue until 100 days after transplantation and immunosuppression is discontinued. If a recipient cannot take oral medications, acyclovir 250 mg/m2IV q12h will be substituted for valacyclovir.  
Notes:
- HSV is an important cause of morbidity after high-dose chemotherapy. Several effective regimens are available. Every recipient receives prophylaxis regardless of HSV serology because of the possibility of primary infection after the test, and the fact that valacyclovir may also prevent VZV reactivation (see below). Our recipients will remain on valacyclovir for a minimum of 6 months.  
- VZV reactivates in a significant proportion of allogeneic HSCT
recipients once antiviral agents are stopped (30-60% in the first year). The morbidity associated with VZV reactivation is high.  
Hence, prophylaxis with antiviral agents is accepted practice.  
Acyclovir (400 mg/d) administered until the end of immunosuppressive therapy has been reported to significantly decrease the incidence of reactivation [115]. It is not known which antiviral and what dose are best to prevent VZV, or for how long to administer it. The universal experience seems to be that reactivation is unusual for as long as patients are on any prophylactic anti-herpesvirus agent [116,117]. We prefer valacyclovir for the convenience of once daily dosing, although it is not FDA-approved for this indication.
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11.1.3.2 cytomegalovirus (cmv)

a) Preemptive Therapy: Recipients with positive pre-transplant serologies for CMV, or whose donors have positive serologies, will undergo weekly monitoring of CMV by polymerase chain reaction (PCR). CMV monitoring by PCR will begin at the time of engraftment after transplantation and continue through day +180. Recipients who continue to require systemic immunosuppression after day +180 will continue to have CMV monitoring performed weekly.  
b) Non-scheduled CMV monitoring may also be warranted for clinical circumstances such as:
- Fever of unknown origin  
- Cytopenias post-transplant
- Pulmonary changes c) If PCR becomes positive, then recipients will be treated with IV ganciclovir or IV
foscarnet according to the following algorithm:  
| Preferred Agents   | Alternatives                       |                  |                            |
|--------------------|------------------------------------|------------------|----------------------------|
| IV GANCICLOVIR     | IV FOSCARNET                       | VALGANCICLOVIR   | CIDOFOVIR                  |
| Induction:         | Induction:                         | Induction:       | Induction:                 |
| 5 mg/kg IV q12h    | 60 mg/kg IV q12h                   | 900 mg PO q12h   | 5 mg/kg IV weekly,  then   |
| for 7 days, then   | for 7 days, then                   | for 7 days, then | maintenance:               |
| maintenance:       | maintenance:                       | maintenance:     | 5 mg/kg every other  week. |
| 5 mg/kg IV QD,     | 60 mg/kg IV QD                     | 900 mg/24h       |                            |
| 5 days/week        | Include probenecid  and hydration. |                  |                            |  
All patients receive one week of induction. Maintenance therapy should continue until one negative or two "low positive" PCR results (henceforth "DNAemia").? Patients with persistent or rising DNAemia during or after induction therapy for CMV will continue or resume ganciclovir at induction doses (5 mg/kg IV q12 hours for 7 consecutive days), because an increase in DNAemia does not indicate ganciclovir-resistant CMV in most patients during or after initial induction If possible, the virus will be sent for sequencing to rule out the presence of mutations associated with ganciclovir or foscarnet resistance (this is not often possible, as the virus must be isolated and grown in culture). If after reinduction there is no improvement, the patient will be changed to another appropriate antiviral therapy (e.g. foscarnet). Otherwise, the patient will be switched to maintenance therapy as described in the table above. If renal adjustment is required for ganciclovir or foscarnet, see http://druginfo.cc.nih.gov or contact unit pharmacist.
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notes

- Valganciclovir and ganciclovir may cause bone marrow suppression. If ANC < 1000:
Start filgrastim (G-CSF) 5-10 µg/kg/d
- Weekly IVIG infusion has no proven benefit in the setting of CMV reactivation without established infection. Therefore, its use will be limited to patients with CMV pneumonitis.
Indications for foscarnet:
- ANC < 500/μl despite G-CSF  
- Platelets < 20,000/mm3
- Resistance to ganciclovir: rising antigenemia after 3 weeks with no response to ganciclovir re-induction.  
11.1.4 Bacterial Pathogens  
11.1.4.1 Hypogammaglobulinemia: Recipients will undergo biweekly monitoring of serum IgG levels beginning prior to transplant and continuing until IgG is > 500 mg/dL for at least 3 consecutive months without supplemental IVIG administration. Recipients with IgG < 400 mg/dL AND a history of repeated sinopulmonary infections (≥ 2 respiratory infections requiring systemic antibiotics over 6 months) will receive IVIG 400-500 mg/kg every 2-4 weeks.
11.1.4.2 Prophylaxis During Chronic GVHD: All recipients receiving treatment for chronic GVHD will receive penicillin V 500 mg PO BID until treatment for chronic GVHD is discontinued. For penicillin-allergic recipients, TMP/SMX 80/400 PO QD can be used. For recipients allergic to both penicillin and sulfa, clarithromycin 500 mg PO QD can be used.
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rationale

There is an increased risk of infection caused by encapsulated organisms, particularly *Streptococcus pneumoniae* in patients with chronic GVHD [119].  
Functional hyposplenism has been considered, and some investigators have attempted to monitor it by ultrasound [120]. Current recommendations support the use of antibiotic prophylaxis with penicillin to prevent these infections. TMP/SMX could be substituted, but only when administered daily.  
11.1.4.3 Immunizations: All recipients will receive immunizations after transplant, according to the following table:
Version Date: September 14, 2018  
| Type                       | 6 Mo                                                                 | 12 Mo   | 14 - 18 Mo   | 24 Mo   |
|----------------------------|----------------------------------------------------------------------|---------|--------------|---------|
| Influenza Virus            | Lifelong, seasonal, starting before HSCT and resuming after 6 months |         |              |         |
| Pneumococcal 7-valent      | x                                                                    | x       |              |         |
| conjugate (0.5 mL, IM)     |                                                                      |         |              |         |
| DTaP a (0.5 mL IM)         | x                                                                    | x       | x            |         |
| Hb conjugate (0.5 mL IM)   | x                                                                    | x       | x            |         |
| IPV < (0.5 mL SQ or IM)    | x                                                                    | x       | x            |         |
| Hepatitis A  (1 mL IM)     | x                                                                    | x       |              |         |
| Hepatitis B  (1 mL IM)     | x                                                                    | x       | x            |         |
| Meningococcal® (0.5 mL SQ) | x                                                                    |         |              |         |
| MR e  (0.5 mL SQ)          | x                                                                    |         |              |         |
| VV (0.5 mL SQ)             | x                                                                    |         |              |         |  
a Diphtheria and Tetanus Toxoids and Acellular Pertussis vaccine, Absorbed (DTaP)
b Haemophilus b Conjugate vaccine c Inactivated Poliovirus vaccine d Meningococcal polysaccharide vaccine (Menomune) is recommended for preadolescents (age 11-12) or other adolescents due to enter high school at 1 year post transplantation. A new meningococcal polysaccharide - protein conjugate vaccine
(Menactra) is also available commercially but it is recommended that this product not be used at this time (Oct 2005) due to reported cases of Guillain-Barre Syndrome linked to the use of Menactra.  
Measles, Mumps, & Rubella vaccine, live, attenuated; only given if patient is immunocompetent at 24 months, i.e., NOT on immune suppression AND NO GVHD for six months.  
Varicella-Zoster Vaccine (VZV); only given if patients are immunocompetent at 24 months, i.e., NOT on immune suppression AND NO GVHD for six months.; If patients were vaccinated, revaccinate ; If patients were never vaccinated with positive serology, DO NOT vaccinate ; If patients were never vaccinated with negative serology, vaccinate; Peds ages 12 mo - 12 y receive a single dose; persons aged ≥ 13 y should receive two doses, 4–8 weeks apart.
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11.2 appendix b management of

A constellation of clinical symptoms and signs has been observed during neutrophil recovery following HSCT, most commonly termed "engraftment syndrome" (ES), but interchangeably termed "capillary leak syndrome" and "autoaggression syndrome". Engraftment syndromes have been observed after both autologous and allogeneic HSCT; in the latter setting, the clinical sequelae of neutrophil recovery were historically attributed to an early, sometimes "hyperacute" graft-versus-host reaction. Our current understanding of ES holds the overproduction of proinflammatory cytokines at the time of neutrophil recovery to be the initiating event. The study of ES has been somewhat hindered by the lack of uniform definition for this clinical entity; therefore, the following criteria have been proposed [105]:  
| Major criteria:   | Fever > 38.3 with no identifiable infectious etiology Erythrodermatous rash involving more than 25% of body  surface area and not attributable to a medication Noncardiogenic pulmonary edema, manifested by diffuse  pulmonary infiltrates and hypoxia            |
|-------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Minor criteria    | Hepatic dysfunction with either total bilirubin > 2 mg/dl or  transaminases levels > two times normal Renal insufficiency (serum creatinine > two times baseline) Weight gain > 2.5% of baseline body weight Transient encephalopathy unexplainable by other causes |  
A diagnosis of ES is established by the presence of all three major criteria, or two major criteria and one or more minor criteria; the clinical signs and symptoms should appear within 96 hours of neutrophil recovery, according to the above proposed definition.
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11.2.2 treatment of engraftment syndrome

The mainstay of therapy for ES is high-dose corticosteroids, based upon the literature on diffuse alveolar hemorrhage [121] in the setting of bone marrow transplantation - a complication that many investigators now believe to be part of the spectrum of ES. Our group has adopted the following treatment schema for patients diagnosed with ES, with satisfactory results:
Day 1: Methylprednisolone 250 mg IV Q6 hours x 4 doses  
| Day 2:      | Methylprednisolone 250 mg IV Q8 hours x 3 doses   |
|-------------|---------------------------------------------------|
| Day 3:      | Methylprednisolone 250 mg IV Q12 hours x 2 doses  |
| Day 4:      | Methylprednisolone 125 mg IV Q12 hours x 2 doses  |
| Day 5:      | Methylprednisolone 60 mg IV Q12 hours x 2 doses   |
| Day 6:      | Methylprednisolone 30 mg IV Q12 hours x 2 doses   |
| Days 7-8:   | Prednisone 60 mg PO QD x 2 days                   |
| Days 9-10:  | Prednisone 50 mg PO QD x 2 days                   |
| Days 11-12: | Prednisone 40 mg PO QD x 2 days                   |
| Days 13-14: | Prednisone 30 mg PO QD x 2 days                   |
| Days 15-16: | Prednisone 20 mg PO QD x 2 days                   |
| Days 17-18: | Prednisone 10 mg PO QD x 2 days                   |
| Day 19:     | Discontinue prednisone                            |  
In the event that symptoms or clinical signs of ES recur during the steroid taper, patients should be retreated with methylprednisolone at a minimum dose of 60 mg IV QD. *This schema is* intended to serve as a guideline and to promote consistency in our clinical practice; it may be modified for individual patients as clinical circumstances warrant.  
11.3 APPENDIX C - GRADING AND MANAGEMENT OF ACUTE GRAFT-VERSUS-HOST DISEASE 11.3.1 Clinical Staging of Acute GVHD [102]  
| Stage   | Skin                    | Liver                      | Gut                     |
|---------|-------------------------|----------------------------|-------------------------|
| +       | Rash < 25% BSA          | Total bilirubin 2-3 mg/dl  | Diarrhea 500-1000ml/d   |
| ++      | Rash 25-50% BSA         | Total bilirubin 3-6 mg/dl  | Diarrhea 1000- 1500ml/d |
| +++     | Generalized erythoderma | Total bilirubin 6-15 mg/dl | Diarrhea >1500ml/d      |
| ++++    | Desquamation and bullae | Total bilirubin > 15 mg/dl | Pain +/- ileus          |  
BSA = body surface area; use "rule of nines" or burn chart to determine extent of rash.
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11.3.2 clinical grading of acute

| Stage                          |            |            |            |     |
|--------------------------------|------------|------------|------------|-----|
| Grade                          | Skin       | Liver      | Gut        | PS  |
| 0 (none)                       | 0          | 0          | 0          | 0   |
| I                              | + to ++    | 0          | 0          | 0   |
| II                             | + to +++   | +          | +          | +   |
| III                            | ++ to +++  | ++ to +++  | ++ to +++  | ++  |
| IV                             | ++ to ++++ | ++ to ++++ | ++ to ++++ | +++ |
| 11.3.3 Treatment of Acute GVHD |            |            |            |     |  
This schema is intended to serve as a guideline and to promote consistency in our clinical practice; it may be modified for individual patients as clinical circumstances warrant.
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grade 0-i gvhd

1) Topical corticosteroids (usually 0.1% triamcinolone; 1% hydrocortisone to face) applied to rash BID.  
Grade II-IV GVHD:
1) Methylprednisolone (MP) 1 mg/kg per dose IV, BID for 4 consecutive days.  
2) If no response after 4 days, continue until response (7-day maximum trial); the dose may be doubled (4 mg/kg/day). 3) If response within 7 days, taper as follows:  
a) 0.75 mg/kg per dose IV BID for 2 days.  
b) 0.5 mg/kg per dose IV BID for 2 days. c) 0.375 mg/kg per dose IV BID for 2 days.  
d) If clinically appropriate, change MP to oral prednisone to equivalent of IV dose) daily for 2 days.  
MP may be converted to prednisone later in the taper at the investigators' discretion.  
e) After this, steroids will be reduced by 10% of starting oral dose each week until a dose of 10 mg/day is reached. Subsequent reductions will be made at the investigators' discretion.  
f) If GVHD worsens during taper, steroids should be increased to previous dose. g) During steroid taper, maintain cyclosporine at therapeutic levels (Section 3.5.1).  
4) If no response is observed within 7 days of MP treatment:
a) Increase Methylprednisolone to 10 mg/kg per dose IV, BID for 2 days. b) If there is no improvement, consideration will be given to using second-line immunosuppressive therapy, e.g., tacrolimus, mycophenolic acid, monoclonal antibodies, or studies of investigational agents for acute GVHD, if they are available.
5) Antifungal prophylaxis with agents effective against mould will be started when it is anticipated that the patient will be receiving steroids at > 1 mg/kg/day of methylprednisolone (or equivalent) for > 2 weeks. (See section 9.1.2.2) Voriconazole, caspofungin, liposomal amphotericin B (Ambisome),
posaconazole or amphotericin B lipid complex (Abelcet) are valid alternatives. During prophylaxis with any of the above agents, fluconazole should be discontinued. In patients with therapeutic cyclosporine levels at the initiation of voriconazole therapy, the cyclosporine or tacrolimus dose should be decreased by approximately 50%. In patients with therapeutic sirolimus levels at the initiation of voriconazole therapy, the sirolimus dose should be decreased by approximately 90%.  
6) Determination of GVHD treatment response should be made within 96 hours of starting the treatment.  
The following are criteria to determine definitions of response to GVHD treatment:
Complete response: Complete resolution of all clinical signs and symptoms of acute GVHD. Partial Response: 50% reduction in skin rash, stool volume or frequency, and/or total bilirubin.  
Maintenance of adequate performance status (Karnofsky Score > 70%).  
Non-responder: < 50% reduction in skin rash, stool volume or frequency, and/or total bilirubin. Failure to maintain adequate performance status (Karnofsky Score  70%).  
Progressive disease: Further progression of signs and symptoms of acute GVHD, and/or decline in performance status after the initiation of therapy.  
11.4 APPENDIX D - NMDP STANDARDS (20TH EDITION) AND NMDP STANDARD POLICIES AND
PROCEDURES
(Please see attached). 11.5 APPENDIX E - NMDP PERIPHERAL BLOOD STEM CELL COLLECTION PROTOCOL (Please see attached).  
| SCORE 0                                                                                                                                                                                                                                   | SCORE 1                                                      | SCORE 2                                                                                                             | SCORE 3                                                                                              |                                                                                                                               |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------|
| PERFORMANCE  SCORE:                                                                                                                                                                                                                       |  Asymptomatic  and fully active  (ECOG 0; KPS or  LPS 100%) |  Symptomatic,  fully ambulatory,  restricted only in  physically strenuous  activity (ECOG 1,  KPS or LPS 80- 90%) |  Symptomatic,  ambulatory,  capable of selfcare, >50% of  waking hours out  of bed (ECOG 2,  KPS or LPS 60- 70%)                                                                                                      |  Symptomatic,  limited self-care, >50% of waking  hours in bed (ECOG  3-4, KPS or LPS  <60%)                                 |
| KPS ECOG LPS SKIN                                                                                                                                                                                                                         |  No Symptoms                                                |  <18% BSA with  disease signs but NO  sclerotic features                                                           |  19-50% BSA  OR involvement  with superficial  sclerotic features  "not hidebound"  (able to pinch) |  >50% BSA OR  deep sclerotic  features "hidebound"  (unable to pinch) OR  impaired mobility,  ulceration or severe  pruritus |
| Clinical features:  Maculopapular  rash  Lichen planus-like  features  Papulosquamous  lesions or ichthyosis  Hyperpigmentation  Hypopigmentation  Keratosis pilaris  Erythema  Erythroderma   Poikiloderma  Sclerotic features |                                                              |                                                                                                                     |                                                                                                      |                                                                                                                               |
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11.6 appendix f - chronic

Pruritus Hair involvement Nail involvement  
| % BSA  involved MOUTH   |  No symptoms   |  Mild symptoms  with disease signs  but not limiting oral  intake significantly   |  Moderate  symptoms with  disease signs with  partial limitation of  oral intake   |  Severe symptoms  with disease signs on  examination with  major limitation of  oral intake   |
|-------------------------|-----------------|------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------|  
| EYES                                              |  No symptoms   |  Mild dry eye  symptoms not  affecting ADL  (requiring eyedrops  < 3 x per day) OR  asymptomatic signs  of  keratoconjunctivitis  sicca   |  Moderate dry  eye symptoms  partially affecting  ADL (requiring  drops > 3 x per day  or punctal plugs),  WITHOUT vision  impairment   |  Severe dry eye  symptoms  significantly  affecting ADL  (special eyeware to  relieve pain) OR  unable to work  because of ocular  symptoms OR loss of  vision caused by  keratoconjunctivitis  sicca   |
|---------------------------------------------------|-----------------|--------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Mean tear test (mm):  >10  6-10  <5  Not done |                 |                                                                                                                                            |                                                                                                                                          |                                                                                                                                                                                                          |  
| SCORE 0                   | SCORE 1                   | SCORE 2                                                                                                                          | SCORE 3                                                                                                                                            |                                                                                                                                                                      |
|---------------------------|---------------------------|----------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| GI TRACT                  |  No symptoms             |  Symptoms such  as dysphagia,  anorexia, nausea,  vomiting, abdominal  pain or diarrhea  without significant  weight loss (<5%) |  Symptoms  associated with  mild to moderate  weight loss (5- 15%)                                                                                |  Symptoms  associated with  significant weight  loss >15%, requires  nutritional  supplement for most  calorie needs OR  esophageal dilation                        |
| LIVER                     |  Normal LFT              |  Elevated  Bilirubin, AP*, AST  or ALT <2 x ULN                                                                                 |  Bilirubin >3  mg/dl or Bilirubin,  enzymes 2-5 x  ULN                                                                                            |  Bilirubin or  enzymes > 5 x ULN                                                                                                                                    |
| LUNGS*                    |  No symptoms             |  Mild symptoms  (shortness of breath  after climbing one  flight of steps)                                                      |  Moderate  symptoms  (shortness of breath  after walking on  flat ground)                                                                         |  Severe symptoms  (shortness of breath  at rest; requiring 02)                                                                                                      |
| FEV1                      |  FEV1 40-59%  OR LFS 6-9 |                                                                                                                                  |                                                                                                                                                    |                                                                                                                                                                      |
|  FEV1 60-79%  OR LFS 3-5 |  FEV1 <39% OR  LFS 10-12 |                                                                                                                                  |                                                                                                                                                    |                                                                                                                                                                      |
|  FEV1 > 80% OR  LFS=2    |                           |                                                                                                                                  |                                                                                                                                                    |                                                                                                                                                                      |
| DLCO JOINTS AND FASCIA    |  No symptoms             |  Mild tightness of  arms or legs, normal  or mild decreased  range of motion  (ROM) AND not  affecting ADL                      |  Tightness of  arms or legs OR  joint contractures,  erythema due to  fasciitis, moderate  decrease ROM  AND mild to  moderate limitation  of ADL |  Contractures  WITH significant  decrease of ROM AND significant  limitation of ADL  (unable to tie shoes,  button shirts, dress  self etc.)                        |
| GENITAL TRACT             |  No symptoms             |  Symptomatic with  mild signs on exam  AND no effect on  coitus and minimal  discomfort with  gynecologic exam                  |  Symptomatic  with moderate  signs on exam  AND with mild  dyspareunia or  discomfort with  gynecologic exam                                      |  Symptomatic  WITH advanced  signs (stricture, labial  agglutination or  severe ulceration)  AND severe pain  with coitus or  inability to insert  vaginal speculum |  
* AP may be elevated in growing children, and not reflective of liver dysfunction Other indicators, clinical manifestations or complications related to cGVHD (check all that apply and assign a score to its severity (0-3) based on its functional impact (none - 0,mild -1, moderate -2, severe - 3)  
| ٱ Esophageal stricture or web___                    | ٱ Pericardial Effusion___       | ٱ Pleural Effusion(s)___         |
|-----------------------------------------------------|---------------------------------|----------------------------------|
| ٱ Ascites (serositis)___                            | ٱ Nephrotic syndrome___         | ٱ Peripheral Neuropathy___       |
| ٱMyasthenia Gravis___                               | ٱ Cardiomyopathy___             | ٱ Eosinophilia > 500μl___        |
| ٱ Polymyositis___                                   | ٱ Cardiac conduction defects___ | ٱ Coronary artery involvement___ |
| ٱ Platelets <100,000/μl ___ٱ Progressive onset ____ | Others_____                     |                                  |  
11.7 APPENDIX G - EXPERIMENTAL TRANSPLANTATION AND IMMUNOLOGY BRANCH
PRECLINICAL SERVICE POLICY FOR SAMPLE HANDLING
11.7.1 Storage/Tracking
Normal donor and patient blood and tissue samples, collected for the purpose of research under IRB approved protocols of the Experimental Transplantation and Immunology Branch, may be archived by the ETIB Preclinical Service. All data associated with archived clinical research samples is entered into the ETIB Preclinical Services' Microsoft Excel databases on frozen cells and plasma. These databases are stored on the NCI group drive in the ETIB Preclinical Service folder. Access to this folder is limited to ETIB clinical staff, requiring individual login and password. All staff in the Preclinical Service laboratory have received annually updated NIH/CIT training and maintain standards of computer security. The data recorded for each sample includes the patient ID, trial name/protocol number, date drawn, treatment cycle/post-transplant time point, cell source (e.g. peripheral blood, lymphapheresis, mobilized peripheral blood stem cells, marrow, pleural fluid) as well as box and freezer location. Patient demographics that correlate treatment outcomes and therapies with the samples can be obtained only through the NCI/ETIB clinical records. As of January 2007, all newly received samples will receive a unique bar code number, which will be added to the sample Preclinical Service database. Only this bar code will be recorded on the sample vial and the vials will not be traceable back to patients without authorized access to the Preclinical Service database. All non-coded samples previously archived will be stripped of identifiers prior to distribution for any use other than as a primary objective of the protocol under which they were collected.  
Samples are stored in locked freezers at -85C (sera and plasma) or under liquid nitrogen (cells),
according to stability requirements. These freezers are located onsite at the Preclinical Service laboratory (12C216) (-85° freezer) or in ETIB common equipment space (CRC/3-3273). Access to samples from a protocol for research purposes will be by permission of the Principal Investigator of that protocol or through his/her submission and IRB approval of the NCI IRB Authorization Form (appended) stipulating whether IRB review is not necessary or IRB approval is granted for the pursuit of this new research activity. All researchers are required to sign a form (attached) stating that the samples are only to be used for research purposes associated with objectives of the original protocol for which the samples were collected, or (using only unlinked or coded samples) for an IRB approved protocol as stipulated on the IRB Authorization Form, and that any unused samples must be returned to the Preclinical Service laboratory.