key: cord-017296-jdp8kgg5
authors: Deschler, Barbara; Waller, Cornelius; Engelhardt, Monika; Müller, Antonia; Luebbert, Michael; Finke, Jürgen; Bertz, Hartmut; Illerhaus, Gerald; Kaskel, Anna-Katharina; Mackensen, A.; Veelken, Hendrik; Rosenthal, F. M.; Müller, Claudia I.; Scheele, Jürgen; Martens, Uwe
title: Particular Treatment Procedures
date: 2008
journal: Concise Manual of Hematology and Oncology
DOI: 10.1007/978-3-540-73277-8_5
sha: 
doc_id: 17296
cord_uid: jdp8kgg5

nan

In preclinical and clinical studies attempts have been made to grow ("expand") cultured stem cells (ex vivo) in order to provide new treatment options. While it is possible to increase cell numbers, relevant stem cell expansion has not been achieved yet. In addition, clinical studies did not demonstrate a significant cost / benefit advantage despite the option of tumor cell depletion.

The following potential areas of use are being evaluated in clinical studies: • Removal of contaminated tumor cells from autologous stem cell grafts • Gene transfer into repopulating stem cells to correct hereditary enzyme deficiencies or for gene marking analyses • Expansion of stem cells and partially differentiated progenitor cells from a single stem cell harvest for repeated clinical use in sequential therapy or tandem transplantation • Expansion of lineage-determined progenitor cells (e.g., myeloid or megakaryopoietic postprogenitor cells) to accelerate hematopoietic regeneration or for differentiation into antigen-presenting dendritic cells • Expansion of bone marrow repopulating stem and progenitor cells from umbilical cord blood samples for transplantation in adult patients • Cell support in allogeneic transplantation • De-/redifferentiation into other organ-type cells ("stem cell plasticity")

• Preparation laboratory (equipped according to national and international guidelines) which allows production of cultivated and gene-transfected cells according to standards • Central filing of all protocols of studies concerned with somatic cell and gene therapy

• Suspension culture using unseparated or CD34 + -separated stem cells (advantage of reduced culture volume). • Addition of recombinant growth factors. Optimal cytokine combinations are currently being tested. Stem cell factor (SCF), FLT3 ligand, thrombopoietin, and IL-11 seem to preserve Part

Hematological treatment aimed to accelerate bone marrow and blood reconstitution by use of autologous peripheral blood hematopoetic stem cells (PBSC) after intense (myeloablative) chemotherapy and / or radiotherapy (total body irradiation, TBI).

In 2004, 22,216 autologous transplantations in Europe (EBMT)

The intensity of conventional chemotherapy is limited, in particular due to hematotoxcity (myelosupression) with neutropenia and thrombocytopenia. Dose-intensive myeloablative therapies require transplantation of hematopoietic stem cells from the patient (autologous transplantation) or a donor (allogeneic transplantation, 7 Chap. 5.3).

• In early studies, hematopoietic stem cells (HSC) were obtained from the bone marrow by aspiration. • Meanwhile, HSC are mainly harvested via blood cell separation (leukapheresis) after stimulation with colony-stimulating factors (CSF) and mobilization into the peripheral blood. Cells are cryopreserved and retransfused after high-dose therapy (peripheral blood stem cells transplantation, PBSCT).

In healthy donors (no stimulation with hematopoietic growth factors, i.e., steady-state conditions), HSCs are a rare population primarily located within the bone marrow. They do not circulate within the peripheral blood without mobilization.

• The number of circulating PBSC increases during the phase of hematopoietic reconstitution after conventional chemotherapy. • The use of hematopoietic growth factors (e.g., granulocyte colony-stimulating factor, G-CSF) after conventional chemotherapy leads to a further increase and is used as a standard method for mobilizing and collecting PBSC. • A sufficient PBSC yield is the prerequisite for high-dose chemotherapy with stem cell transplantation (PBSCT).

• Identification of hematopoietic progenitor cells by detection of CD34 antigen expression (1-4% of mononuclear cells in the bone marrow or mobilized blood are CD34-positive; however, recent data have shown the existence of CD34-negative hematopoietic stem cells, characterized by CD133, Thy-1, Oct-4, c-kit/CD117, SP phenotype).

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• Over 90% of these cells are "committed progenitor cells" which have lost the ability to renew themselves. Only pluripotent stem cells hold this feature and have the potential for complete hematopoietic reconstitution (7 Chap. 1.3 ). • In mice, sufficient numbers of pluripotent stem cells for complete hematopoietic reconstitution can be attained with 100 highly purified cells. In humans after myeloablative chemotherapy (conditioning), 2-4 × 106 CD34-positive cells/kg body weight (BW) are regarded as sufficient. Optimal reconstitution of all cell lineages is achieved by transplantation of ≥ 4 × 106 CD34-positive cells/kg BW.

Patients who are candidates for high-dose chemotherapy with peripheral blood stem cell transplantation (PBSCT) should initially be treated with conventional chemotherapy in order to combine maximum clinical response with early PBSC mobilization and harvesting. Myelosuppression generated by PBSC mobilizing chemotherapy should ideally be brief (without affecting the stem cell compartment) and have a maximum effect on the underlying disease:

• So far, there is no optimal mobilization protocol which could be used for the entire range of cancer patients. Most commonly, cyclophosphamide is used to facilitate PBSC mobilization, e.g., in the VCP-E Protocol (Etoposide (VP16), Cyclophosphamide, Cisplatin, and Epirubicin 7 Protocol 13.1.1) followed by administration of recombinant human G-CSF. • Administration of rh G-CSF increases the number of circulating multipotent progenitor cells by factor 10. Maximum PBSC mobilization occurs concomitantly with an increase in neutrophil granulocytes after the leukocyte nadir has been passed.

PBSC are harvested as an outpatient procedure when the leukocyte count (WBC) is > 5,000-10,000/µl and CD34 + cells are > 10-20/µl blood, using a standard cell separator ("leukapheresis"). The procedure is well tolerated. Possible electrolyte imbalances can be compensated.

• In patients without prior chemo-or radiotherapy, sufficient numbers of PBSC can usually be harvested with 1-2 leukaphereses. • For reasons of quality control, volume, differential leukocyte count, percentage of CD34-positive cells, viability, and sterility of every stem cell apheresis sample must be determined. • Leukapheresis PBSC may be further processed (e.g., CD34-positive or -negative selection 7 Chap. 5.1) or directly preserved in liquid nitrogen (at −196°C) . Cryopreserved cells can be stored for many years prior to transplantation. • Both processing and storage of the cell samples are carried out under GMP conditions (Good Manufacturing Practice, EU GMP guidelines) in accordance with national laws.

Randomized studies have shown high-dose chemotherapy to be advantageous in the treatment of high-grade (and indolent) non-Hodgkin's and Hodgkin's lymphoma compared with standarddose chemotherapy. EBMT (European Group for Blood and Marrow Transplantation) has published guidelines for autologous stem cell transplantation as follows:

• In certain patient groups with high-grade non-Hodgkin's lymphomas (NHL), Hodgkin's disease, multiple myeloma, and chemosensitive relapses of germ cell tumors, high-dose chemotherapy with autologous transplantation is effective and regarded as standard of care. • In solid tumors (e.g., sarcoma, breast cancer, or ovarian carcinoma), high-dose chemotherapy should only be carried out within clinical studies. • High-dose chemotherapy with stem cell support has been proven to be a potentially effective treatment for various other malignancies. However, the clinical benefit as compared to standard treatment has to be established in randomized clinical trials for each individual disease entity.

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• Bone marrow aplasia: high-dose chemotherapy causes bone marrow aplasia, which is overcome within approximately 10 (granulocytes) to 14 days (thrombocytes) after autologous PBSC transplantation. Infections and hemorrhage may occur within this period, and most Co: Co:

patients require antibiotics and blood products for 4-8 days. Fungal infections are rare. Viral infections may occur due to reactivation (HSV, VZV, rarely CMV). • Gastrointestinal toxicity: oropharyngeal mucositis, gastroenteritis • Pulmonary toxicity: with use of certain cytostatics (e.g., busulfan, cyclophosphamide, thiotepa, BCNU), inflammatory changes (fibrosis, alveolar hemorrhage, infection), pulmonary edema, pulmonary damage, and "acute respiratory distress syndrome" (ARDS) • Cardiotoxicity: cardiac damage due to cytostatics, e.g., cyclophosphamide (cardiac insufficiency, transmural hemorrhagic myocardial necrosis), anthracyclines (acute and chronic cardiotoxicity); cardiac complications may be intensified in cases of preceding radiotherapy or anthracycline treatment • Renal dysfunction: renal insufficiency or acute renal failure due to cytostatic drugs, antibiotic treatment with aminoglycosides, insufficient hydration during treatment, tumor lysis, and blood pressure fluctuations; renal insufficiency is usually reversible • Hepatic dysfunction: besides fully reversible short-term increases in hepatic enzymes, rare occurrences of veno-occlusive disease (VOD) 

Progress in PBSCT is to be expected in the following areas:

• Sequential transplantation (e.g., multiple myeloma) • Use of new hematopoietic growth factors for stimulation of recovery of platelets and neutrophils to further shorten the cytopenic phase after chemotherapy • Improved management of side effects of induction therapy • Generation of immunocompetent cells for the treatment of minimal residual disease 3.

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. Autologous Transplantation

Transfer of pluripotent hemato-/ lymphopoietic stem cells from healthy donors to recipients.

• Bone marrow transplantation (BMT)

• Peripheral blood stem cell transplantation (PBSCT) • Umbilical cord blood transplantation (UCBT)

In 2004, 7407 allogeneic transplantations were performed in Europe (EBMT).

The success of allogeneic transplantation is based on two therapeutic principles which distinguish this method from conventional chemotherapy and autologous transplantation:

• Conditioning: immuno-and myeloablative high-dose chemotherapy and/or total body irradiation (TBI) • Graft versus leukemia effect (GVL effect): immunological reaction of donor lymphocytes from the graft against malignancy in the recipient.

While cytostatic treatment largely reduces the malignant clone, the GVL effect seems to ensure long-term reduction of the relapse rate. Complete T-cell depletion of the graft leads to increased relapse rates. 

Donor Search Strategy 1. HLA-Typing of the patient and close family (siblings, parents) 2. In urgent cases, parallel search for donors among members of the extended family and unrelated donors

Of particular relevance are HLA alleles of the classes I A, I B and II DR.

• In patients with related donors, one allele difference in graft versus host direction (GVH direction) and three allele differences in host versus graft direction (HVG direction) are acceptable. This configuration provides for equally successful transplantation results as complete HLA identity. • Class II DR alleles should be identified using genetic typing methods (DNA typing).

• The relevance of the class II loci DQ and DP is not yet known. When several HLA-A-, B-, and DR-identical donors are available, further selection criteria are considered (sex, age, CMV status, blood group).

In the typing of unrelated donors, 8 HLA antigens (two alleles each of A, B, DRB1*, DQB1*) of the patient and the prospective unrelated donor are important. Recently, the relevance of HLA-C has been emphasized, especially for graft rejection and NK cell-induced GVL effects. DP seems to be of little importance in relation to alloreactions.

• DR typing should be based on high-resolution DNA typing (4 digits), just as serological HLA-A, -B, and -C typing should be replaced by DNA methods (2 digits; recently 4 digits). • Particularly in younger patients, minor differences may be acceptable. • Additional HLA-DQB1* and/or HLA-DPB1* differences are at present no reason for exclusion of a donor. Allele differences in the A or B locus as well as minor DRB1* differences may also be acceptable, especially in patients with an aggressive underlying disease. • Due to its low predictive value, mixed lymphocyte culturing (MLC) is no longer a routine procedure. • The donor search should be coordinated by recognized immunogenetics laboratories together with a transplantation center.

Options in case of unsuccessful donor search:

• Transplantation from donor after T-cell depletion • Transplantation of umbilical cord blood (UCB): of particular importance in pediatric patients and young adults.

Cord blood banks are currently being established. So far, over 2,000 UCB transplants have been carried out worldwide. If necessary, several HLA differences may be accepted, especially in pediatric patients. The main limiting factor is the cell content of the graft in relation to the patient's body weight.

Hematopoietic stem cells are characterized by their expression of specific surface markers (CD34, CD133). The following hematopoetic stem cell compartments are used for transplantation:

• Bone marrow (BM) → bone marrow transplantation (BMT)

• Peripheral blood stem cells (PBSC, after stimulation with G-CSF) → peripheral blood stem cell transplantation ((PBSCT) • Cryopreserved umbilical cord blood stem cells (UCB) → UCB transplantation (UCBT)

Normally, the freshly collected grafts are transplanted without being manipulated or cryopreserved. However, in specific situations, the graft needs to be modified:

Treatment Procedures

• T-cell depletion: prevention of graft versus host reactions • Selection of CD34-positive cells ("stem cell selection"): reduction of immune reactions and elimination of malignant cells (autologous transplantation) (→ 7 Chap. 5.1)

Bone marrow is collected under general anesthesia (approximately 60 min) by bilateral puncture and aspiration from multiple sites of the iliac crest.

• The bone marrow is anticoagulated and can be stored without cryopreservation for up to 1 day without significant stem cell loss (duration of transport with unrelated donor transplants). • Potential disadvantages for the donor include blood loss, local pain and hematomas and side effects related to anesthesia.

Recently, peripheral blood stem cells have increasingly been used for allogeneic transplantation.

• Pretreatment of the donor with 5-24 µg/kg/day G-CSF s.c. for 4-6 days.

• Stem cell harvest by one or more leukaphereses.

• Depending on the rate of yield, the graft may contain more CD34 + stem cells than comparable bone marrow products as well as 10 times more CD3 + T-lymphocytes. • In randomized studies, allogeneic transplantation of peripheral blood hematopoetic cells conferred an increased risk of GVHD compared with allogeneic BMT. • Hematopoietic engraftment is faster with PBSC than with bone marrow. • PBSC grafts allow manipulation (stem cell enrichment, selective T-cell depletion, "graft engineering"), facilitating GVHD prophylaxis and transplantation despite HLA barriers.

Successful allogeneic transplantation is ultimately "myeloablative, " which is a result of both the conditioning therapy and the donor T-lymphocytes. Side effects:

• Toxic side effects of the conditioning therapy (chemotherapy, radiotherapy) depending on the therapy protocol • Infections during the phase of bone marrow aplasia (until bone marrow reconstitution): bacterial infections, fungal infections (Candida, Aspergillus), viral infections (CMV, HSV) • Long-term consequences: pulmonary fibrosis, bronchiolitis obliterans, gonadal insufficiency, hormonal deficiencies, cardiomyopathy, cataract, secondary neoplasia

• Lymphocytes in the graft play a major role in "alloreactions, " i.e., immunological reaction of transplanted immunologically active donor cells versus recipient organism → inflammatory immunological reactions in immunogenic structures, such as skin (dermatitis), intrahepatic bile ducts (cholestasis), and intestinal epithelium (enteritis); in extreme cases: destruction. • The period of immunological adaptation and development of tolerance is divided into an acute phase (100 days after transplantation, acute GVHD) and a chronic phase (3-12 months after transplantation, chronic GVHD). • Acute GVHD especially affects the skin, liver, and intestine. • Chronic GVHD can potentially affect any organ. Particularly skin and mucosal dryness, generalized sicca syndrome with conjunctivitis, malabsorption syndrome, chronic cholestasis, weight loss, and increasing pulmonary obstruction may occur. In extreme cases, sclerodermatype skin symptoms and other autoimmune disorders may develop. • GVHD prophylaxis with immunosuppressive drugs (e.g., cyclosporin A + methotrexate)

is usually required in the initial weeks and months following transplantation. Unlike transplanted solid organs, an immune system developing from transplanted stem cells gradually becomes tolerant of the recipient. • Especially in the first few months of this process, the patient is at risk of immune reactions and opportunistic infections (CMV, VZV, PCP, pneumococcal pneumonia) → close monitoring by the transplantation center in collaboration with the referring physician. A high degree of patient compliance is required.

Since different transplantation units may use center-specific protocols, consultation with experienced centers is always advisable. Variations may exist in relation to the following:

• Conditioning: based on high-dose chemotherapy with or without total body irradiation (IBI) • Management of immunosuppression • Stem cell graft engineering / manipulation Furthermore, new protocols with less intensive conditioning regimens are currently being developed, for different indications and entry criteria (age). In protocols with reduced conditioning regimens, the age limit for allogeneic transplantation from related or unrelated donors has been raised from 55 years to approximately 70 years.

See Table ( p. 308)

Remission-inducing therapy (transplantation in complete remission generally leads to improved long-term outcome and cure rates)

• Chemotherapy ± radiotherapy, high-dose or intensity-reduced (see below)

• Transplantation: transplantation of BM, PBSC, or cord blood from healthy donors via intravenous infusion; engraftment of hematopoietic stem cells in the bone marrow

• Supportive administration of antibiotics, erythrocytes and platelets during the neutropenic (10-15 days) and thrombocytopenic phase (12-25 days) • Parenteral nutrition and pain therapy in cases of severe mucositis (5-15 days). Mucositis prevention (palifermin) • Prophylaxis and, if necessary, treatment of graft versus host disease (GVHD), (cyclosporine levels). Monitoring for clinical signs of GVHD, exanthema, diarrhea, icterus, dryness of the mouth, conjunctivitis, mucositis • Rehabilitation, outpatient follow-up, return to work after approximately 4-9 months 

Terms such as "mini-transplantation, " "micro-transplantation, " or "non-myeloablative transplantation" have been used to describe reduced-intensity conditioning protocols which are based on dose modifications of traditional regimens (containing TBI 10-13.2 Gy or busulfan 16 mg/kg). However, these protocols represent a variation of allogeneic SCT with otherwise similar procedures and comparable immunological and infection-related problems (GVHD, opportunistic infections). Due to reduced acute toxicity, these regimens may preferably be used in patients with additional comorbidities or in older patients.

• The immunotherapeutic benefit of the GVL effect was demonstrated in patients with relapse of CML (7 Chap. 7.3.1) after allogeneic bone marrow transplantation: the transfer of immunologically active donor lymphocytes resulted in complete remission in > 50% of these patients. • So-called myeloablative therapy (e.g., with 10-14 Gy TBI or busulfan 16 mg/kg in combination with cyclophosphamide) is an aggressive conditioning regimen with multiple side effects limiting use to younger patients without significant comorbidity. Older patients therefore do not equally benefit from allogeneic transplantation with standard conditioning regimens. • Dose escalation studies with radiotherapy or chemotherapy in patients with aggressive, therapy-refractory leukemias have shown that despite excessive toxicity and mortality, the risk of relapse after allogeneic transplantation was not significantly reduced. • Recent animal experiments have shown that stable lympho-hematopoietic engraftment of donor cells can be achieved with significantly lower irradiation doses of 2 Gy, causing less side effects (so-called immunoconditioning). Parallel to the GVL effect, the treatment induces "donor chimerism, " i.e., simultaneous existence of lymphatic and hematopoietic cells of the donor and the recipient. • "Allogeneic transplantation with reduced conditioning" is aimed at utilizing the GVL effect without the disadvantages of maximum tolerable conditioning treatment. This therapeutic approach eventually constitutes a form of immunotherapy based on T-cell-mediated cytotoxicity.

• Initial clinical studies, including patients over 60 years of age, have demonstrated the feasibility of this therapeutic approach. In individual cases, post-therapeutic complete and partial remission has been described. Its applicability in patients of > 60 years of age allows curative therapeutic attempts, particularly in patients with AML, MDS, and low grade NHL. • Donor chimerism of lymphatic and hematopoietic cells without significant myelosuppression (leuko-or thrombocytopenia) was achieved. • Other approaches use fludarabine in combination with alkylating agents to increase tolerance prior to allogeneic transplantation. Fludarabine has a particularly toxic effect on T-cells and has added benefit in the treatment of lymphomas. • The long-term outcome (overall survival) after reduced-intensity conditioning has not been established yet. Randomized clinical trials for individual disease entities are necessary. (7 Chap. 7 .2), ALL acute lymphatic leukemia (7 Chap. 7.1.1) , CLL chronic lymphatic leukemia, MPS myeloproliferative syndrome (7 Chap. 7 .3), NHL non-Hodgkin's lymphomas (7 Chap. 7.5) , OMF osteomyelofibrosis, CR complete remission, PR partial remission Experimental treatment procedure. Transfusion of donor granulocytes with the aim of correcting neutropenia (e.g., after chemotherapy).

The use of rhG-CSF for mobilization of donor granulocytes has been a prerequisite for successful development of granulocyte transfer. Several phase I/II studies have produced promising results in neutropenic patients with severe infections. Prospective phase III studies with comparison of granulocyte transfusion vs. standard of care are still pending.

• Donors and recipients must be ABO and Rh compatible (granulocyte concentrates contain erythrocytes). CMV status has to be considered • Stimulation and mobilization of granulocytes with rhG-CSF (5 µg/kg) s.c.

• After 12 h, leukapheresis → with HAES 6% (to accelerate sedimentation) and sodium citrate (anticoagulant) • Granulocyte yield depends on donor WBC count, leukapheresis efficiency, and volume of processed blood • Irradiation of granulocyte concentrates with 30 Gy to avoid graft versus host reaction (large numbers of lymphocytes and stem cells in the leukapheresis sample) • Store concentrates at room temperature, without agitation for up to 24 hrs.

• Carry out transfusion as soon after collection as possible • Therapeutic success depends primarily on the number of transfused cells → transfusion of ≥ 1.5 × 108 granulocytes per kg body weight of recipient

Clinical studies in patients with severe infections not responding to anti-infective treatment, with concomitant neutropenia (< 500 neutrophils/µl) and without foreseeable bone marrow recovery. . Granulocyte Transfusion

• Direct pulmonary toxicity, dyspnea, hypoxemia → monitor O2 saturation • Transfusion-induced acute pulmonary insufficiency (TRALI: transfusion-related acute lung injury) (7 Chap. 9.8) • Alloimmunization against HLA class I antigens and granulocyte-specific antigens → inefficiency of further transfusions, fever, respiratory symptoms, anaphylactic reactions

• Inclusion in clinical study, information and signed consent of donor and recipient • ABO and Rh compatible donor, check CMV serology of donor and recipient (no transfusion from CMV-positive donor to CMV-negative recipient)

• Clinical diagnosis: case history, physical examination • Laboratory tests: full blood count with differential, blood group, hepatic and renal function parameters, coagulation parameters, serology (HAV, HBV, HCV, CMV, HIV, Treponema pallidum); female patients: pregnancy test where appropriate • Cross-match (blood group testing) before each granulocyte transfusion • ECG, optional chest x-ray, abdominal sonography (splenic enlargement) • G-CSF 5 µg/kg s.c., 12 h before each leukapheresis • Prior to each new leukapheresis as well as 5 and 30 days after the final donation: blood count with differential, urea and electrolytes, serum creatinine, bilirubin, ALT

• Leukapheresis sample irradiated with 30 Gy • Administer as soon as possible (within 6 h)

• Serological (erythrocytic) and leukocytic (lymphocyte toxicity test) compatibility must be assessed prior to each granulocyte transfusion. After administration of amphotericin B, wait at least 6 h before giving a granulocyte transfusion (pulmonary toxicity). • Ten minutes prior to transfusion, premedication with antihistamines and antipyretics (e.g., paracetamol). • Recommended transfusion rate: 1 × 1010 cells per hour. Use standard filters (pore size 170-230 µm). • Monitor blood pressure, pulse, respiratory rate, and O2 saturation from the beginning until 1 h after transfusion. • Evaluate transfusion success by measuring the post-transfusion granulocyte increase. Specific or non-specific modulation of the immune system with the objective of immunologically mediated destruction of malignant cells.

Immunotherapy approaches to cancer treatment have been studied since the nineteenth century. Four different approaches can be distinguished:

• Active specific immunotherapy • Active non-specific immunotherapy • Passive immunotherapy • Adoptive immunotherapy

The T-cellular immune response is crucial to recognize and eliminate tumor cells. T-cell subtypes include:

A more recent classification is based on the cytokines produced by T-lymphocytes. CD4-positive T-lymphocytes are subclassified as:

T-lymphocytes do not recognize intact proteins, but peptides bound to MHC (major histocompatibility complex) molecules. Different types of proteins can be processed by intracellular proteases, the resulting peptides are bound to MHC molecules and expressed on the cell surface of antigen-presenting cells. Consequently, T-cell recognition is not limited to surface markers, but may include intracellular antigens, thus multiplying the diversity of T-cell target epitopes. Prerequisite for the recognition of peptides is the binding to MHC molecules:

• Peptides binding to MHC I: 7-14 amino acids • Peptides binding to MHC II: 14-24 amino acids

The specifics of antigen recognition by T-lymphocytes are dependent on the interaction of the variable region of the T-cell receptor (TCR) molecule with the MHC-peptide complex. This binding site of the TCR is coded by a unique gene segment, which is formed by the recombination of a V-D-J segment for the β locus and a V-J segment for the α locus during T-cell differentiation. This combinatorial diversity is amplified by the random addition of nucleotides and is the basis for the diversity of the T-cell repertoire.

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. Immunotherapy

Proteins (e.g. melanoma antigens tyrosinase, MAGE 1-3, Melan-A) are processed by intracellular proteases. Resulting peptides are bound by MHC molecules and presented at the cell surface. T-cell receptor (TCR) binds to MHC-peptide-complex. ER Endoplasmatic Reticulum

The binding of a TCR to a specific peptide-MHC complex alone is not sufficient for activation and proliferation of naive T-cells. Additional signals are required:

• Adhesion molecules which facilitate contact with the targeted cell • Costimulatory signal: costimulatory molecules are the antigens of the B7-family (B7-1, B7-2), which are expressed by antigen-presenting cells (APC). "Professional APCs" play a central role for the initiation of the immune response. The antigens interact with suitable ligands on T-cells (CD28, CTLA-4). If a naive T-cell meets a non-professional APC (e.g., tumor cell presenting a peptide matching a specific TCR), a second costimulatory activation signal is missing. The result is the induction of anergy, i.e., the T-cell is refractory to further stimulatory signals.

The immune system of a tumor patient is rarely capable of inducing regression of manifest tumors and metastases. This observation supports the hypothesis, that tumor specific antigens lead to incomplete activation of the immune system, or tumor cells "escape" immunologic toxicity through induction of immunosuppression. Various "tumor escape" mechanisms of neoplastic cells have been described:

• Lack of expression of costimulatory molecules on tumor cells • Loss or downregulation of MHC molecules (β2-microglobulin, HLA-A or -B) or receptors for apoptosis • Loss of transport proteins (TAP) → reduced presentation of tumor peptides with MHC molecules • Selection of so-called antigen-loss variants, without expression of tumor-associated antigens (MAGE, tyrosinase, gp100) • Induction of angiogenetic or antiapoptotic factors • Secretion of immunoinhibitory cytokines, such as TGF-β and IL-10, by tumor cells

Dendritic cells or monocytes (professional antigen-presenting cells, APC) ingest antigenic material and disintegrate and process it to peptides. Peptides are attached to MHC-I or MHC-II molecules and transported to the cell membrane where they are presented. The TCR binds to peptide-MHC complexes, and cellular inter-action is supported by adhesion molecules and costimulatory signals such as B7 (on APC) and CD28 (T-cells). Depending on the antigen and the cytokine environment, cell types preferably induced are Th2 or Th1 helper cells. Th2 cells produce cytokines (such as , which are particularly important for the stimulation and differentiation of B-cells to antibody-producing plasma cells. Th2 cells also interact with B-cells, which can ingest and process the same antigen complex (e.g., bacterium, tumor cells) with the help of membrane antibody molecules or the B-cell receptor -similar to dendritic cells. The interaction of B-and T-cells leads to a coordinated antigen-specific B-and T-cell immune response. Th1 cells produce interferon-γ and IL-2, which particularly promote the maturation of MHC-I-peptide complex specific cytotoxic T-cells

The term "active non-specific immunostimulation" relates to the administration of modifiers, which can directly modulate the immune system. "Non-specific" indicates the lack of antigen specificity. Non-specific immunity is mainly based on activated macrophages, but also NK cells and neutrophils. The following are possible biological response modifiers: . Immunotherapy Treatment of a disease by expression of one / multiple specific genes in a cell or group of cells. The production of the desired gene product corrects the genetic defect or alters cellular function.

• Somatic gene therapy: expression of genes in differentiated somatic cells • Germline therapy: expression of genes in fertilized human oocytes or embryonic stem cells

Adequate methods of gene transfer are a basic requirement for gene therapeutic approaches. In most cases, so-called vectors are used to transport the therapeutic gene construct into the target cells. Due to directed genetic deletions, viral vectors are usually unable to replicate after transfection of the target cell. Viral vectors are produced with the help of "packaging cells, " which provide the necessary structural proteins for replication. After transfection of the genetic construct, these cells produce the required vector.

The following are important criteria for the evaluation of gene transfer methods:

• Efficacy: transfection quality (transient or stable), transfection efficiency, tropism (potential of organ-specific gene transfers), biological efficacy (expression of gene products) • Safety: tolerance, adverse effects, immunogenicity • Production effort, cost and compliance with GMP / GLP / GCP criteria

The first somatic gene therapy was carried out in September 1990 at the NIH, USA, on a 4-year-old girl with adenosine deaminase (ADA) deficiency. Since then, more than 630 clinical gene therapy studies have been licensed worldwide and 3,500 patients have been treated. Besides monogenic hereditary diseases, gene therapy is used particularly in patients with advanced malignancy, AIDS, or multifactorial diseases such as coronary heart disease or rheumatoid arthritis.

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. Gene Therapy

Gene Therapy

• Induction of specific immune responses by transfer of immunostimulating genes (e.g., interleukin or interferon genes) into tumor cells, bystander cells, or immunoeffector cells • Transfer of tumor suppressor genes → correction of regulation defects in tumor proliferation • Blockade of oncogenic effects via antisense strategies • Transfer of "suicide genes" into tumor cells: suicide genes usually encode enzymes (e.g., HSV thymidine kinase), which by means of phosphorylation convert non-toxic prodrugs (e.g., ganciclovir) into toxic substances, thus selectively killing HSV-TK-expressing cells • Transfer of cytostatic drug resistant genes (MDR-1, aldehyde dehydrogenase, O6-alkylguanine-DNA alkyltransferase, cytidine deaminase) into hematopoietic stem cells to increase the in vivo resistance of the hematopoietic system to cytostatic drugs and alleviate the hematotoxicity of subsequent chemotherapy Until now, mainly critically ill patients with short life expectancy were included in gene therapy studies. Therefore, the results of these studies focus primarily on safety and side effects, rather than on curative aspects. Initial data on the safety of gene therapy methods showed that close surveillance is required:

• After administration of high doses of adenoviral vectors, immune reactions to adenoviral proteins and pulmonary toxicity were detected in patients with cystic fibrosis. • Severe adverse reactions also occurred during a clinical trial in patients with the X-chromosomal form of severe combined immunodeficiency (X-SCID), carried out in Paris. Newborn children suffering from this so far incurable disease were cured by transfer of the normal gene. However, approximately 3 years after treatment, 3 out of 11 children developed T-cell leukemia, the cause of which seemingly involved the insertion of the retroviral vector. The exact causes of this severe adverse effect are under intense medical and molecular biological investigation. • A 17-year-old patient with a severe congenital metabolic disease died in September 2000 in the USA after infusion of high doses of genetically modified adenovirus into the liver artery.

The importance of strict adherence to the highest standards for production and quality control of gene therapy drugs as well as the conduct of controlled clinical trials was underlined by these cases.

At present, gene therapy is used in clinical studies only (7 Chap. 3.7) . Besides legal aspects mentioned above, national and international guidelines and regulation for gene therapy products and studies have to be followed.

Target entities

• Infections / parasitic diseases 13%

• Cardiovascular diseases 10%

Therapeutic approaches (number of trials)

• Immunotherapy 16

• Tumor suppressor regulation 9

• Proof-of-concept has been established for the biological or clinical efficacy of gene transfer in studies:

• Induction or amplification of tumor-specific immune responses in tumor vaccination studies • Occasional tumor regression or stable disease after transfer of tumor suppressor genes (p53) • Decreased incidence of GVHD after allogeneic hematopoietic transplantation due to transfer of HSV-TK suicide genes into allogeneic donor lymphocytes after administration of ganciclovir • Correction of the immunodeficiency in X-chromosomal severe combined immunodeficiency (X-SCID) • In patients suffering from hemophilia, factor VIII use was reduced by 50-80% after intramuscular injection of AAV vectors carrying the wildtype gene Furthermore, genetic marking of hematopoietic stem cells showed that while contributing to long-term hematopoietic reconstitution, transplanted stem cell products can potentially contain malignant cells which act as a starting point for relapse. Although this method is a diagnostic procedure, the results of these studies are seminal for future advancement of transplantation strategies and trends in gene therapy with respect to the hematopoietic system. . Inhibition of Angiogenesis

In 1970, Folkman described the transition of solid tumors from an avascular resting state to a vascularized phase with optimal tumor oxygenation and nutrition. Only in the vascularized state, i.e., after the "angiogenic switch", accelerated tumor proliferation, metastasis, and generalization can occur, as for example in prostate carcinoma, breast cancer, and renal cell carcinoma. Similarly, increased bone marrow microvessel density has been described in proliferating hematological neoplasia, particularly in patients with leukemia (AML, ALL, CML) and myelodysplasia.

Inhibition of tumor-induced angiogenesis and the angiogenic switch of human tumors (transition from avascular state to vascularized proliferating tumor) was first demonstrated as an effective means of treatment with the VEGF-receptor antibody bevacizumab (7 Chap. 3.5 compound has been approved for treatment of metastatic colorectal cancer and non-small cell lung cancer (NSCLC). Similar approaches are followed with small molecule inhibitors of angiogenesis, e.g. sorafenib and sunitinib (7 Chap. 3.6) . Increasing understanding of the molecular mechanisms of cancer and hematologic malignancies led to new strategies in the development of therapeutic agents, and this has resulted in the introduction of new drugs such as tyrosine kinase inhibitors in the treatment of malignant diseases. Promising new approaches include: • RNA-targeted therapy • Aurora kinase inhibition • HSP (heat shock proteins) as targets for cancer therapeutics • Telomerase therapeutics

In recent years the members of the RNA family have grown rapidly. In addition to the coding messenger RNAs (mRNAs) and transcriptional RNAs [ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs)], another subfamily, called small RNAs, has been discovered, each member of which has its own particular function. Small RNAs do not code for proteins, but instead control the transcription and translation of protein-coding RNAs. The small RNA subfamily contains small interfering RNAs (siRNAs), microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), and small nuclear RNAs (snRNAs). siRNAs and miRNAs have attracted much attention due to their potential diagnostic and therapeutic applications in different diseases.

siRNAs and miRNAs are generated using the same pathway by processing long doublestranded RNA (dsRNA) or microRNA precursors with an endonuclease known as "Dicer". Subsequently, the RNAs attach to an RNA-induced silencing complex (RISC) and are directed to the messenger RNA (mRNA) of interest which is marked for cleavage or inhibition of translation.

Developmental Therapeutics

These RNAs are 21-24 nucleotides in length, double-stranded, and have 3' overhangs of 2 nucleotides. siRNAs mediate the phenomenon of RNA interference (RNAi) which is a pathway for silencing the transcript of an active gene. RNAi, discovered in 1998, is now a standard laboratory tool for knocking down gene expression.

Exogenous synthetic siRNAs or endogenously expressed siRNAs attach to an RNA-induced silencing complex (RISC) and are directed to the messenger RNA (mRNA) of interest which is marked for destruction.

The highly specific silencing effect of RNAi has emerged as an attractive way to allow specific inhibition of the function of any chosen target genes, including those involved in diseases such as cancer, AIDS, and hepatitis.

• The biggest obstacle to the development of RNAi-based therapeutics is the delivery. Trigger RNAs (dsRNAs from which siRNAs are derived by the action of Dicer) can be expressed from vectors or delivered as artificial siRNAs. Inserting foreign vector sequences (gene therapy) into chromosomal DNA includes the problem of insertional activation and inactivation of cellular genes. Direct administration of siRNAs would require siRNAs that are stable and modified to be resistant to nucleases. • Drug therapies using siRNAs are now in clinical trials for treating age-related macular degeneration and respiratory syncytial virus infection (RSV).

MicroRNAs are short 20-22 nucleotide RNA molecules that are negative regulators of gene expression in a variety of eukaryotic organisms. miRNAs are involved in numerous cellular processes including development, differentiation, proliferation, apoptosis, and stress response. About 350 miRNAs have been identified in humans, with the total predicted to eventually reach 1,000 or more. miRNA mutations or altered expression correlate with various human cancers and indicate that miRNAs can function as tumor suppressors or oncogenes ("oncomirs"). Like siRNAs, miRNAs are generated from long primary precursor RNAs before being processed by the Dicer protein in the cytoplasm and incorporated as mature miRNA into the RNA-induced silencing complex (RISC). Whereas siRNAs perfectly match with the target mRNA, most miRNAs do not match the target sequence exactly, enabling them to bind to multiple mRNAs. Instead of destruction of mRNA as in RNAi interference, imprecise matching results in inhibition of translation.

About half of the annotated human miRNAs map within fragile regions of chromosomes in cancer genomes. Expression profiling of about 200 miRNAs has been shown to be a more accurate method of classifying cancer subtypes than using the expression of protein-coding genes.

Gene therapies using miRNAs might be an effective approach to restore tumor suppressor function or to block oncogene activation.

• Administration of synthetic antisense oligonucleotides that encode sequences that are complementary to mature oncogenic miRNAs-termed anti-miRNA oligonucleotides (AMOs)might effectively inactivate miRNAs in tumors and slow their growth. . Developmental Therapeutics Part Treatment Procedures

• Antagomirs, a novel class of chemically engineered oligonucleotides appear to be specific and effective silencers of miRNA expression in mice when conjugated with cholesterol.

The serine-threonine kinases Aurora A, B, and C represent a family of mitotic regulators which are essential for mitotic progression, spindle formation, centrosome maturation, chromosomal segregation, and cytokinesis. Selectively inhibiting Aurora kinase activity by RNAi or small molecules leads to chromosome segregation errors and deregulation of the spindle checkpoint associated with cell death.

Aurora A localizes to centrosomes / spindle poles and is required for spindle assembly, whereas Aurora B is a chromosome passenger protein required for phosphorylation of histone H3, chromosome segregation, and cytokinesis. Elevated expression of Aurora A and B has been detected in many human cancers and overexpression of Aurora A has been shown to induce oncogenic transformation in vitro.

Tumor cells treated with Aurora kinase inhibitors show normal timing of expression of core cell cycle regulators, such as cyclins, and do not undergo arrest or delay transit through mitosis as classic antimitotic agents do. The antiproliferative effect is unique, in that tumor cells, especially those lacking functional p53 damage response, are catastrophically driven forward and out of an aberrant mitosis, which finally leads to cell death due to massive chromosomal instability.

Although Aurora A has received most of the attention so far in terms of a link with human cancer, Aurora B might be the more suitable anticancer drug target, because inhibition of Aurora B rapidly results in a catastrophic mitosis.

• Small molecule inhibitors in development: Hesperadin, AZD1152 (phase I), MK0457(= VX-680, inhibits also Flt-3, phase I), MLN8054 (Aurora A, phase I), JNJ-7706621 (inhibits also cyclin-dependent kinases, CDKs) • Aurora kinases are only expressed and active as kinases during mitosis, therefore, it is assumed that non-proliferating cells would not be adversely affected by Aurora kinase inhibitors • Reduction or abrogation of histone H3 phosphorylation could serve as biomarker • MK0457(= VX-680) is effective against imatinib-and dasatinib-resistant bcr-abl(T315I) kinase in vitro

The heat shock protein (HSP) family of proteins has emerged as a target for cancer drug discovery because it is important for mediating the action of oncogenically relevant growth factor receptors and their downstreaming signaling elements.

Many HSPs form multimolecular complexes that act as chaperones binding other proteins, denoted as client proteins. HSP90 consists of two isoforms and is one of the most abundant cellular chaperone proteins. It is a cellular chaperone required for refolding of denatured proteins, cellular survival under stress conditions, and the maturation of a subset of proteins that play key roles in oncogenesis. Therefore, HSP90 does not catalyze a single reaction, but mediates the stability and function of multiple client proteins such as: Natural products, including the ansamycin antibiotic geldanamycin and radiciol, that bind selectively to HSP90 and inhibit its chaperone function have been identified. However, geldanamycin is limited by its hepatotoxicity for clinical use, but a less toxic derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) has been identified.

• Due to poor chemical stability and bioavailability subsequent geldanamycin-and non-geldanamycin-based compounds are in development: KOS-953 (Tanespimycin, cremaphor-based formulation of 17-AAG, phase I/II), KOS-1022 (17-DMAG, Alvespimycin hydrochloride, orally active, phase I), CNF1010 (oil-in-water nanoemulsion of 17-AAG, phase I), CNF2024 (orally, phase I), IPI-504 (water soluble, phase I), SNX-5542 (orally active, preclinical). • Tumors which are dependent on a given client protein are particularly sensitive to degradation of HSP90 inhibitors. The proteins observed to be most sensitive to HSP90 inhibitor-induced degradation are the HER2 and MET receptor tyrosine kinases, RAF-1 kinase, and the estrogen and androgen receptors. • HSP90 inhibitors might be particularly effective in cancer cells in which Rb is mutationally inactivated (e.g., small cell lung cancer). Typically, HSP90 inhibition induces cell cycle arrest in G1 phase. However, in Rb-defective cells, tumor cells fail to arrest in G1 and enter a mitotic block with disordered prometaphase and unstable kinetochore assembly which is followed by apoptotic cell death. • HSP90 inhibitors enhance the activity of cytotoxics including taxanes, anthracyclines, hormonal agents, bortezomib, trastuzumab, and radiation. There is a schedule dependence in context with an intact retinoblastoma (Rb) function due to its growth arrest in G1 phase of the cell cycle. • Inhibitors of angiogenesis may also sensitize tumor cells to HSP90 inhibitors, because hypoxic tumor cells are under greater stress and HIF1alpha is also a client protein required for survival under these conditions.

Maintenance of telomeres at the ends of chromosomes is essential for unlimited cellular proliferation and confers immortality in cancer cells. Since most cancer cells are reliant on telomerase for their survival, this enzyme represents an attractive mechanism-based target for the development of new cancer therapeutics.

Telomeres consist of repetitive double-stranded repeats of the sequence TTAGGG associated with telomere-binding proteins. Their major function is to cap the ends of chromosomes and to provide genetic stability. Telomerase is a ribonucleoprotein enzyme, which synthesizes telomere repeats de novo. In human cells, the telomerase holoenzyme consists of a high-molecular weight complex with a template-containing RNA subunit, hTR, and protein components including the catalytic subunit human telomerase reverse transcriptase, hTERT. In addition, several additional molecules might play a role in regulating in vivo activity of telomerase such as the chaperone HSP90/p32. In most normal somatic cells telomerase activity is absent and telomere repeats are lost with cell division and with ageing. Telomere attrition beyond a certain threshold is assumed to uncap chromosome ends which subsequently induces DNA damage and onset of replicative senescence. In contrast, about 80-90% of cancer cells have detectable telomerase activity, which leads to stabilization of telomeres and unlimited growth potential.

Strategies targeting telomeres / telomerase in cancer cells:

• Oligonucleotide antagonists against hTR or hTERT (e.g., GRN163L, a thio-phosphoramidate oligonucleotide targeting the template region of hTR as a "template antagonist"; phase I/II). Like siRNA therapeutics (see above) there remains the issue of delivery and stability of antisense oligonucleotides. • Small molecule inhibitors of the catalytic component hTERT (e.g., BIBR1532; preclinical); the antiproliferative effect is not induced by inhibition of the enzyme itself but through consecutive telomere dysfunction. The lag period of telomere shortening limits the widespread use of this approach. • Heat shock protein 90 (HSP90) inhibitors which compromise telomerase assembly by targeting HSP90/p23 (phase II) 

Dose intensity and the toxicity and efficacy of allogeneic hematopoietic cell transplantation

Allogeneic stem-cell transplantation from related and unrelated donors in older patients with myeloid leukemia

The graft-versus-lymphoma-effect: clinical review and future opportunities

Hematopoietic stem-cell transplantation

How I treat refractory acute GVHD

Results of the EBMT activity survey 2005 on haematopoietic stem cell transplantation: focus on increasing use of unrelated donors

Adult umbilical cord blood transplantation

Ref: Web: Web: . Allogeneic Hematopoetic Stem Cell Transplantation

Side effects of retroviral gene transfer into hematopoietic stem cells

Gene therapy strategies in prostate cancer

Good manufacturing practice production of adenoviral vectors for clinical trials

Regulatory considerations for novel gene therapy products: a review of the process leading to the first clinical lentiviral vector

A review of gene therapy for haematological disorders

Survival of the fittest: in vivo selection and stem cell gene therapy

Angiogenesis: formation of new blood vessels; mostly formation of new capillaries from pre-existing blood vessels. Inhibition of angiogenesis: through inhibition of endogenous angiogenic factors or administration of physiological / pharmacological angiogenesis inhibitors

Physiological angiogenesis is essential for the development of embryonic organs as well as the regulation of the adult vascular system: • Embryogenesis: vasculogenesis, i.e., formation of new angioblast-derived blood vessels • Proliferation of uterine epithelia, menstruation • Proliferation and vascularization of muscle tissue • Wound healing, bone growth, nerve regeneration, hair growth • Regulation of vascular permeability → homeostasis In adult organisms, angiogenesis is typically strictly regulated and of limited duration

VE-cadherin • Fibroblast growth factors (aFGF, bFGF), hepatocyte growth factor (HGF) • Platelet-derived growth factor

• Transforming growth factors (TGFα, TGFβ), tumor necrosis factor alpha (TNFα) • Interleukin

• Integrins α v β3, α v β5, α5β1 • Prostaglandins E1 (PgE1) and E2 (PgE2) • Matrix metalloproteinases (MMPs)

Angiogenesis in health and disease

Use of angiogenesis inhibitors in tumour treatment

The biology of VEGF and its receptors

Role of angiogenesis in tumor growth and metastasis

Angiogenesis and leukemia

The pathophysiologic role of VEGF in hematologic malignancies: therapeutic implications

Angiogenesis-dependent diseases and angiogenesis therapy

Disrupting tumour blood vessels

Ref: Web: Web: . Inhibition of Angiogenesis

Such folding is incompatible with telomerase function and may induce rapid telomere uncapping. The toxicity of such molecules is not yet clarified

• Immunotherapy with vaccines targeting hTERT-specific epitopes on cancer cells (GV1001; PrimoVax and TeloVax trial for pancreatic cancer, phase II)

Telomerase-directed gene therapy: − Suicide gene therapy: the hTERT promoter is linked to a proapoptotic gene or cytotoxic prodrug

Oncolytic viral therapy: viral genes which are critical for replication are placed under control of the hTERT gene promoter. This results in virus vectors that are replicated only in telomerase-positive cells, and then spread to adjacent cells on cell lysis

Heat shock protein 90 modulators in hematologic neoplasms

The major world of microRNAs. Horizon Symposia

Inhibition of drug resistant mutants of Abl, Kit, and EGF receptor kinases

siRNAs: applications in functional genomics and potential as therapeutics

Oncomirs-microRNAs with a role in cancer

Aurora-kinase inhibitors as anticancer agents

Overcoming the immortality of tumour cells by telomere and telomerase based cancer therapeutics: current status and future prospects

Silencing of microRNAs in vivo with "antagomirs"

Telomerase therapeutics for cancer: challenges and new directions

Heat shock protein 90 as therapeutic target in solid tumors

Therapeutic potential of RNA interference

Treatment with monoclonal antibodies directed against tumor antigens. Mechanisms of tumor cell lysis by monoclonal antibodies: • Antibody-dependent cellular cytotoxicity; ADCC • Complement-dependent cytotoxicity; CDC • Intrinsic cytotoxic activity / induction of apoptosis • Carrier of a cytotoxic substance (toxins, radionuclides, cytostatics) • Antibody variants: murine antibodies, chimeric / humanized antibodies, bispecific antibodies, immunotoxins / radioconjugates Indications ( 

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