Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Allogeneic Stem Cell Transplantation

Writer and Curator: Larry H. Bernstein, MD, FCAP

This article has the following structure:

9.3.1  Cell based immunotherapy

9.3.2  Photodynamic therapy (PDT)

9.3.3  Small molecules targeted therapy drugs; Tyrosine kinase inhibitors; imatinib (Gleevec/Glivec) and gefitinib (Iressa).

9.3.4 Graft versus Host Disease

9.3.5 Aspergillus Complicating Allogeneic Transplantation

Introduction

9.3.1 Allogeneic Stem Cell Treatment

http://www.lls.org/treatment/types-of-treatment/stem-cell-transplantation/allogeneic-stem-cell-transplantation

Allogeneic stem cell transplantation involves transferring the stem cells from a healthy person (the donor) to your body after high-intensity chemotherapy or radiation.

Allogeneic stem cell transplantation is used to cure some patients who:

• Are at high risk of relapse
• Don’t respond fully to treatment
• Relapse after prior successful treatment

Allogeneic stem cell transplantation can be a high-risk procedure. The high-conditioning regimens are meant to severely or completely impair your ability to make stem cells and you will likely experience side effects during the days you receive high-dose conditioning radiation or chemotherapy. The goals of high-conditioning therapy are to:

treat the remaining cancer cells intensively, thereby making a cancer recurrence less likely
inactivate the immune system to reduce the chance of stem cell graft rejection
enable donor cells to travel to the marrow (engraftment), produce blood cells and bring about graft versus tumor effect

Possible Adverse Effects

The immune system and the blood system are closely linked and can’t be separated from each other. Because of this, allogeneic transplantation means that not only the donor’s blood system but also his or her immune system is transferred. As a result, these adverse effects are possible:

• Immune rejection of the donated stem cells by the recipient (host-versus-graft effect)
• Immune reaction by the donor cells against the recipient’s tissues (graft-versus-host disease [GVHD])

The immune reaction, or GVHD, is treated by administering drugs to the patient after the transplant that reduce the ability of the donated immune cells to attack and injure the patient’s tissues. See Graft Versus Host Disease.

Allogeneic stem cell transplants for patients who are older or have overall poor health are relatively uncommon. This is because the pre-transplant conditioning therapy is generally not well tolerated by such patients, especially those with poorly functioning internal organs. However, reduced intensity allogeneic stem cell transplants may be an appropriate treatment for some older or sicker patients.

T-Lymphocyte Depletion

One goal of allogeneic stem cell transplant is to cause the T lymphocytes in the donor’s blood or marrow to take hold (engraft) and grow in the patient’s marrow. Sometimes the T lymphocytes attack the cancer cells. When this happens, it’s called graft versus tumor (GVT) effect (also called graft versus cancer effect). The attack makes it less likely that the disease will return. This effect is more common in myeloid leukemias than it is in other blood cancers.

Unfortunately, T lymphocytes are the same cells that cause graft versus host disease (GVHD). Because of this serious and sometimes life-threatening side effect, doctors in certain cases want to decrease the number of T lymphocytes to be infused with the stem cells. This procedure, called T-lymphocyte depletion, is currently being studied by researchers. The technique involves treating the stem cells collected for transplant with agents that reduce the number of T lymphocytes.

The aim of T-lymphocyte depletion is to lessen GVHD’s incidence and severity. However, it can also cause increased rates of graft rejection, a decreased GVT effect and a slower immune recovery. Doctors must be careful about the number of T lymphocytes removed when using this technique.

Stem Cell Selection

Stem cell selection is another technique being studied in clinical trials that can reduce the number of T lymphocytes that a patient receives. Because of specific features on the outer coat of stem cells, doctors can selectively remove stem cells from a cell mixture. This technique produces a large number of stem cells and fewer other cells, including T lymphocytes.

9.3.2 Defining Characteristics of  Stem Cells

http://stemcells.nih.gov/info/basics/pages/basics1.aspx

Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth. In addition, in many tissues they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell.

Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and replace worn out or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions.

Until recently, scientists primarily worked with two kinds of stem cells from animals and humans: embryonic stem cells and non-embryonic “somatic” or “adult” stem cells. The functions and characteristics of these cells will be explained in this document. Scientists discovered ways to derive embryonic stem cells from early mouse embryos more than 30 years ago, in 1981. The detailed study of the biology of mouse stem cells led to the discovery, in 1998, of a method to derive stem cells from human embryos and grow the cells in the laboratory. These cells are called human embryonic stem cells. The embryos used in these studies were created for reproductive purposes through in vitro fertilization procedures.

When they were no longer needed for that purpose, they were donated for research with the informed consent of the donor. In 2006, researchers made another breakthrough by identifying conditions that would allow some specialized adult cells to be “reprogrammed” genetically to assume a stem cell-like state. This new type of stem cell is called induced pluripotent stem cells (iPSCs).

Stem cells differ from other kinds of cells in the body. All stem cells—regardless of their source—have three general properties: they are capable of dividing and renewing themselves for long periods; they are unspecialized; and they can give rise to specialized cell types.

Stem cells are capable of dividing and renewing themselves for long periods. Unlike muscle cells, blood cells, or nerve cells—which do not normally replicate themselves—stem cells may replicate many times, or proliferate. A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells. If the resulting cells continue to be unspecialized, like the parent stem cells, the cells are said to be capable of long-term self-renewal.

Scientists are trying to understand two fundamental properties of stem cells that relate to their long-term self-renewal:

1. Why can embryonic stem cells proliferate for a year or more in the laboratory without differentiating, but most adult stem cells cannot; and
2. What are the factors in living organisms that normally regulate stem cell proliferation and self-renewal?

Discovering the answers to these questions may make it possible to understand how cell proliferation is regulated during normal embryonic development or during the abnormal cell division that leads to cancer.

Stem cells are unspecialized. One of the fundamental properties of a stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. For example, a stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell), and it cannot carry oxygen molecules through the bloodstream (like a red blood cell). However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells.

Stem cells can give rise to specialized cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation. While differentiating, the cell usually goes through several stages, becoming more specialized at each step. Scientists are just beginning to understand the signals inside and outside cells that trigger each step of the differentiation process. The internal signals are controlled by a cell’s genes, which are interspersed across long strands of DNA and carry coded instructions for all cellular structures and functions. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment. The interaction of signals during differentiation causes the cell’s DNA to acquire epigenetic marks that restrict DNA expression in the cell and can be passed on through cell division.

Adult stem cells typically generate the cell types of the tissue in which they reside. For example, a blood-forming adult stem cell in the bone marrow normally gives rise to the many types of blood cells. It is generally accepted that a blood-forming cell in the bone marrow—which is called a hematopoietic stem cell—cannot give rise to the cells of a very different tissue, such as nerve cells in the brain.

Through years of experimentation, scientists have established some basic protocols or “recipes” for the directed differentiation of embryonic stem cells into some specific cell types (Figure 1). (For additional examples of directed differentiation of embryonic stem cells, refer to the NIH stem cell report available at

http://stemcells.nih.gov/info/scireport/pages/2006report.aspx.)

http://stemcells.nih.gov/StaticResources/images/figure1_sm.jpg

9.3.3 Types of Stem Cell Transplants for Treating Cancer

http://www.cancer.org/treatment/treatmentsandsideeffects/treatmenttypes/bonemarrowandperipheralbloodstemcelltransplant/stem-cell-transplant-types-of-transplant

In a typical stem cell transplant for cancer very high doses of chemo are used, often along with radiation therapy, to try to destroy all the cancer cells. This treatment also kills the stem cells in the bone marrow. Soon after treatment, stem cells are given to replace those that were destroyed. These stem cells are given into a vein, much like a blood transfusion. Over time they settle in the bone marrow and begin to grow and make healthy blood cells. This process is called engraftment.

There are 3 basic types of transplants. They are named based on who gives the stem cells.

• Autologous (aw-tahl-uh-gus)—the cells come from you
• Allogeneic (al-o-jen-NEE-ick or al-o-jen-NAY-ick)—the cells come from a matched related or unrelated donor
• Syngeneic (sin-jen-NEE-ick or sin-jen-NAY-ick)—the cells come from your identical twin or triplet

Autologous stem cell transplants

These stem cells come from you alone. In this type of transplant, your stem cells are taken before you get cancer treatment that destroys them. Your stem cells are removed, or harvested, from either your bone marrow or your blood and then frozen. To find out more about that process, please see the section “What’s it like to donate stem cells?” After you get high doses of chemo and/or radiation the stem cells are thawed and given back to you.

One advantage of autologous stem cell transplant is that you are getting your own cells back. When you donate your own stem cells you don’t have to worry about the graft attacking your body (graft-versus-host disease) or about getting a new infection from another person. But there can still be graft failure, and autologous transplants can’t produce the “graft-versus-cancer” effect.

This kind of transplant is mainly used to treat certain leukemias, lymphomas, and multiple myeloma. It’s sometimes used for other cancers, like testicular cancer and neuroblastoma, and certain cancers in children.

Getting rid of cancer cells in autologous transplants

A possible disadvantage of an autologous transplant is that cancer cells may be picked up along with the stem cells and then put back into your body later. Another disadvantage is that your immune system is still the same as before when your stem cells engraft. The cancer cells were able to grow despite your immune cells before, and may be able to do so again. The need to remove cancer cells from transplants or transplant patients and the best way to do it is being researched.

Doing 2 autologous transplants in a row is known as a tandem transplant or a double autologous transplant. In this type of transplant, the patient gets 2 courses of high-dose chemo, each followed by a transplant of their own stem cells. All of the stem cells needed are collected before the first high-dose chemo treatment, and half of them are used for each transplant. Most often both courses of chemo are given within 6 months, with the second one given after the patient recovers from the first one.

Allogeneic stem cell transplants

In the most common type of allogeneic transplant, the stem cells come from a donor whose tissue type closely matches the patient’s. (This is discussed later under “HLA matching” in the section called “ Donor matching for allogeneic transplant.”) The best donor is a close family member, usually a brother or sister. If you do not have a good match in your family, a donor might be found in the general public through a national registry. This is sometimes called a MUD (matched unrelated donor) transplant. Transplants with a MUD are usually riskier than those with a relative who is a good match.

Blood taken from the placenta and umbilical cord of newborns is a newer source of stem cells for allogeneic transplant. Called cord blood, this small volume of blood has a high number of stem cells that tend to multiply quickly. But the number of stem cells in a unit of cord blood is often too low for large adults, so this source of stem cells is limited to small adults and children. Doctors are now looking at different ways to use cord blood for transplant in larger adults, such as using cord blood from 2 donors.

Pros of allogeneic stem cell transplant: The donor stem cells make their own immune cells, which could help destroy any cancer cells that remain after high-dose treatment. This is called the graft-versus-cancer effect. Other advantages are that the donor can often be asked to donate more stem cells or even white blood cells if needed, and stem cells from healthy donors are free of cancer cells.

Cons to allogeneic stem cell transplants: The transplant, also known as the graft, might not take — that is, the donor cells could die or be destroyed by the patient’s body before settling in the bone marrow. Another risk is that the immune cells from the donor may not just attack the cancer cells – they could attack healthy cells in the patient’s body. This is called graft-versus-host disease (described in the section called “Problems that may come up shortly after transplant”). There is also a very small risk of certain infections from the donor cells, even though donors are tested before they donate. A higher risk comes from infections you have had, and which your immune system has under control. These infections often surface after allogeneic transplant because your immune system is held in check (suppressed) by medicines called immunosuppressive drugs. These infections can cause serious problems and even death.

Allogeneic transplant is most often used to treat certain types of leukemia, lymphomas, multiple myeloma,myelodysplastic syndrome, and other bone marrow disorders such as aplastic anemia.

Mini transplants (non-myeloablative transplants)

For some people, age or certain health conditions make it more risky to wipe out all of their bone marrow before a transplant. For those people, doctors can use a type of allogeneic transplant that’s sometimes called a mini-transplant. Compared with a standard allogeneic transplant, this one uses less chemo and/or radiation to get the patient ready for the transplant. Your doctor might refer to it as a non-myeloablative transplant or mention reduced-intensity conditioning (RIC). The idea here is to kill some of the cancer cells along with some of the bone marrow, and suppress the immune system just enough to allow donor stem cells to settle in the bone marrow.

Unlike the standard allogeneic transplant, cells from both the donor and the patient exist together in the patient’s body for some time after a mini-transplant. But slowly, over the course of months, the donor cells take over the bone marrow and replace the patient’s own bone marrow cells. These new cells can then develop an immune response to the cancer and help kill off the patient’s cancer cells — the graft-versus-cancer effect.

Syngeneic stem cell transplants – for those with an identical sibling

This is a special kind of allogeneic transplant that can only be used when the recipient has an identical sibling (twin or triplet) who can donate — someone who will have the same tissue type. An advantage of syngeneic stem cell transplant is that graft-versus-host disease will not be a problem. There are no cancer cells in the transplant, either, as there would be in an autologous transplant.

A disadvantage is that because the new immune system is so much like the recipient’s immune system, there is no graft-versus-cancer effect, either. Every effort must be made to destroy all the cancer cells before the transplant is done to help keep the cancer from relapsing (coming back).

9.3.4 Graft versus Host Disease

http://bethematch.org/For-Patients-and-Families/Life-after-transplant/Graft-versus-host-disease–GVHD-/

Graft-versus-host disease(GVHD) occurs because of differences between the cells of your body and the donated cells and is a common side effect of an allogeneic bone marrow transplant.

An allogeneic transplant uses blood cells from a family member, unrelated donor or cord blood unit. GVHD can affect many different parts of the body including the skin, eyes, mouth, stomach, and intestines.

There are two types of GVHD:

• Acute GVHD: Develops in the first 100 days or so after transplant but can occur later. This primarily affects the skin, stomach, intestines, and liver.
• Chronic GVHD: Usually develops 3-6 months after transplant, but signs can appear earlier or later. If you have had or currently have acute GVHD, you are more likely to have chronic GVHD.

The severity of acute and chronic GVHD can range from mild to life-threatening.

Doctors often see mild GVHD as a good thing after an allogeneic transplant when the transplant was done for a blood cancer. It is a sign that the donor’s immune system is working to destroy any remaining cancer cells. Patients who experience some GVHD have a lower risk of the cancer returning after transplant than patients who do not develop GVHD. If the transplant was to treat a disease other than cancer disease, like aplastic anemia, then the doctor may want to treat even mild GVHD.

Graft-versus-Host Disease

JLM Ferrara, JE Levine, P Reddy, and E Holler
Lancet. 2009 May 2; 373(9674): 1550–1561.
http://dx.doi.org:/10.1016/S0140-6736(09)60237-3

The number of allogeneic hematopoietic cell transplantations (HCT) continues to increase with more than 25,000 allogeneic transplantations performed annually. The graft-versus-leukemia / tumor (GVL) effect during allogeneic HCT effectively eradicates many hematological malignancies.¹ The development of novel strategies that use donor leukocyte infusions, non-myeloablative conditioning and umbilical cord blood (UCB) transplantation have helped expand the indications for allogeneic HCT over the last several years, especially among older patients.² Improvements in infectious prophylaxis, immunosuppressive medications, supportive care and DNA-based tissue typing have also contributed to improved outcomes after allogeneic HCT.¹ Yet the major complication of allogeneic HCT, graft-versus-host disease (GVHD), remains lethal and limits the use of this important therapy.² Given current trends, the number of transplants from unrelated donors is expected to double within the next five years, significantly increasing the population of patients with GVHD. In this seminar we review advances made in identifying the genetic risk factors and pathophysiology of this major HCT complication, as well as its prevention, diagnosis and treatment.

Etiology and Clinical Features

Fifty years ago Billingham formulated three requirements for the development of GVHD: the graft must contain immunologically competent cells; the recipient must express tissue antigens that are not present in the transplant donor; and the recipient must be incapable of mounting an effective response to eliminate the transplanted cells.³ We know now that the immunologically competent cells are T cells, and that GVHD can develop in various clinical settings when tissues containing T cells (blood products, bone marrow, and solid organs) are transferred from one person to another who is not able to eliminate those cells.^4, 5 Patients, whose immune systems are suppressed, and who receive white blood cells from another individual, are at particularly high risk for GVHD.

GVHD occurs when donor T cells respond to genetically defined proteins on host cells. The most important proteins are Human Leukocyte Antigens (HLA)^2, 6, 7, which are highly polymorphic and are encoded by the major histocompatibility complex (MHC). Class I HLA (A, B, and C) proteins are expressed on almost all nucleated cells of the body at varying densities. Class II proteins (DR, DQ, and DP) are primarily expressed on hematopoietic cells (B cells, dendritic cells, monocytes), but their expression can be induced on many other cell types following inflammation or injury. High-resolution DNA typing of HLA genes with polymerase chain reaction (PCR)-based techniques have now largely replaced earlier methods. The incidence of acute GVHD is directly related to the degree of mismatch between HLA proteins^8, 9 and thus ideally, donors and recipients are matched at HLA-A, -B, -C, and -DRB1, (“8/8 matches”), but mismatches may be tolerated for UCB grafts (see below).^10–12

Non-HLA Genetics

Despite HLA identity between a patient and donor, approximately 40% of patients receiving HLA-identical grafts develop acute GVHD due to genetic differences that lie outside the HLA loci, or “minor” histocompatibility antigens (HA). Some minor HAs, such as HY and HA-3, are expressed on all tissues and are targets for both GVHD and GVL.¹³ Other minor HAs, such as HA-1 and HA-2, are expressed most abundantly on hematopoietic cells (including leukemic cells) and may therefore induce a greater GVL effect with less GVHD.^13, 14

Polymorphisms in both donors and recipients for cytokines that are involved in the classical `cytokine storm’ of GVHD (discussed below) have been implicated as risk factors for GVHD.¹⁵ Tumor Necrosis Factor (TNF)-α, Interleukin 10 (IL-10), Interferon-γ (IFNγ) variants have correlated with GVHD in some, but not all, studies.^16–18 Genetic polymorphisms of proteins involved in innate immunity, such as nucleotide oligomerization domain 2 and Keratin 18 receptors, have also been associated with GVHD.^19–22 Future strategies to identify the best possible transplant donor will probably incorporate both HLA and non-HLA genetic factors.

Clinical Features of Acute GVHD

Based on an early Seattle experience, acute GVHD was defined to occur prior to day 100, whereas chronic GVHD occurred after that time.^23–25 This definition is far from satisfactory, and a recent National Institutes of Health classification includes late-onset acute GVHD (after day 100) and an overlap syndrome with features of both acute and chronic GVHD.²⁶ Late-onset acute GVHD and the overlap syndrome occur with greater frequency after reduced-intensity conditioning (RIC), an increasingly widespread technique (see below). As shown in Table 1, the clinical manifestations of acute GVHD occur in the skin, gastrointestinal tract and liver.²⁷ In a comprehensive review, Martin et al found that at the onset of acute GVHD, 81% of patients had skin involvement, 54% had GI involvement, and 50% had liver involvement.²³ Recent data suggest that lungs might also be targets of experimental GVHD.²⁸

Acute GVHD Symptoms

Table 1

Pathophysiology of Acute GVHD

Two important principles are important to consider regarding the pathophysiology of acute GVHD. First, acute GVHD reflects exaggerated but normal inflammatory mechanisms mediated by donor lymphocytes infused into the recipient where they function appropriately, given the foreign environment they encounter. Second, the recipient tissues that stimulate donor lymphocytes have usually been damaged by underlying disease, prior infections, and the transplant conditioning regimen.²⁹ As a result, these tissues produce molecules (sometimes referred to as “danger” signals) that promote the activation and proliferation of donor immune cells.^42–45 Mouse models havebeen central to our identification and understanding of the pathophysiologic mechanisms of GVHD, and canine models have been critical to the development of clinically useful strategies for GVHD prophylaxis and treatment and to the development of donor leukocyte infusions.^36, 46, 47 Based largely on these experimental models, the development of acute GVHD can be conceptualized in three sequential steps or phases: (1) activation of the APCs; (2) donor T cell activation, proliferation, differentiation and migration; and (3) target tissue destruction (Figure 3).

Figure 3

GVHD Pathophysiology

In Phase I, the recipient conditioning regimen damages host tissues and causes release of inflammatory cytokines such as TNFα, IL-1 and IL-6. Increased levels of these cytokines leads to activation of host antigen presenting cells (APCs). In Phase II, host APCs activate mature donor cells. The subsequent proliferation and differentiation of these activated T cells produces additional effectors that mediate the tissue damage, including Cytotoxic T Lymphocytes, Natural Killer (NK) cells, TNFα and IL-1. Lipopolysaccharide (LPS) that has leaked through the damaged intestinal mucosa triggers additional TNFα production. TNFα can damage tissue directly by inducing necrosis and apoptosis in the skin and GI tract through either TNF receptors or the Fas pathway. TNFα plays a direct role in intestinal GVHD damage which further amplifies damage in the skin, liver and lung in a “cytokine storm.”

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2735047/bin/nihms-115970-f0003.jpg

Phase I: Activation of Antigen Presenting Cells (APCs)

The first step involves the activation of APCs by the underlying disease and the HCT conditioning regimen. Damaged host tissues respond by producing “danger” signals, including proinflammatory cytokines (e.g., TNF-α), chemokines, and increased expression of adhesion molecules, MHC antigens and costimulatory molecules on host APCs.^42, 48–50 A recent report demonstrated that at one week after HCT, increased levels of TNF-α receptor I, a surrogate marker for TNF-α, strongly correlated with the later development of GVHD.⁵¹ Damage to the GI tract from the conditioning is particularly important because it allows for systemic translocation of additional inflammatory stimuli such as microbial products including lipopolysaccaride (LPS) or other pathogen-associated molecular patterns that further enhance the activation of host APCs.⁴⁹ The secondary lymphoid tissue in the GI tract is likely the initial site of interaction between activated APCs and donor T cells.⁵² These observations have led an important clinical strategy to reduce acute GVHD by reducing the intensity of the conditioning regimen. Experimental GVHD can also be reduced by manipulating distinct subsets of APCs.^53,54 In addition, non-hematopoietic stem cells, such as mesenchymal stem cells or stromal cells, can reduce allogeneic T cell responses, although the mechanism for such inhibition remains unclear.²

The concept that enhanced activation of host APCs increases the risk for acute GVHD unifies a number of seemingly disparate clinical associations with that risk, such as advanced stages of malignancy, more intense transplant conditioning regimens and histories of viral infections. APCs detect infections by recognizing conserved molecular patterns that are unique to microbes, called pathogen-associated molecular patterns (PAMPs). Among the classes of receptors that recognize such patterns, the Toll-like receptors (TLR) are the best characterized.⁵⁵ For example, TLR4 recognizes LPS⁵⁵ and mice with mutant TLR4 receptors that do not respond to LPS cause less GVHD when used as donors.⁵⁶ Other TLRs that recognize viral DNA or RNA also activate APCs and may enhance GVHD, providing a potential mechanistic basis for increased GVHD associated with viral infections such as cytomegalovirus (CMV).⁵⁷

Phase II: Donor T Cell Activation

The core of the GVH reaction is Step 2, where donor T cells proliferate and differentiate in response to host APCs. The “danger” signals generated in Phase I augment this activation at least in part by increasing the expression of costimulatory molecules.⁵⁸ Blockade of co-stimulatory pathways to prevent GVHD is successful in animal models, but this approach has not yet been tested in large clinical trials.²

In mouse models, where genetic differences between donor and recipient strains can be tightly controlled, CD4⁺ cells induce acute GVHD to MHC class II differences, and CD8⁺ cells induce acute GVHD to MHC class I differences.^59–61 In the majority of HLA-identical HCTs, both CD4⁺ and CD8⁺ subsets respond to minor histocompatibility antigens and can cause GVHD in HLA-identical HCT.

Regulatory T cells can suppress the proliferation of conventional T cells and prevent GVHD in animal models when added to donor grafts containing conventional T cells.⁶² In mice, the Foxp3 protein functions as a master switch in the development of regulatory T cells, which normally constitute 5% of the CD4+ T cell population.⁶² Regulatory T cells secrete anti-inflammatory cytokines IL-10 and Transforming Growth Factor(TGF)-β and can also act through contact-dependent inhibition of APCs.⁶² It is likely that the use of regulatory T cells in clinical acute GVHD will require improved techniques to identify and expand them.

Natural Killer T cell (NKT) 1.1⁺ subsets of both the host and donors that have been shown to modulate acute GVHD.⁶³ Host NKT cells have been shown to suppress acute GVHD in an IL-4 dependent manner.⁶⁴ A recent clinical trial of total lymphoid irradiation used as conditioning significantly reduced GVHD and enhanced host NKT cell function.⁶⁵ By contrast, donor NKT cells can reduce GVHD and enhance perforin mediated GVL in an experimental model.⁶⁶

Activation of immune cells results in rapid intracellular biochemical cascades that induce transcription of genes for many proteins including cytokines and their receptors. Th1 cytokines (IFN-γ, IL-2 and TNF-α) are produced in large amounts during acute GVHD. IL-2 production by donor T cells remains the principal target of many current clinical therapeutic and prophylactic approaches to GVHD, such as cyclosporine, tacrolimus and monoclonal antibodies (mAbs) directed against IL-2 and its receptor.⁹ But emerging data indicate an important role for IL-2 in the generation and maintenance of CD4⁺ CD25⁺ T regs, suggesting that prolonged interference with IL-2 may have an unintended consequence of preventing the development of long term tolerance after allogeneic HCT.⁶⁷ IFN-γ has multiple functions and can either amplify or reduce GVHD.^68,69 IFN-γ may amplify GVHD by increasing the expression of molecules such as chemokines receptors, MHC proteins, and adhesion molecules; it also increases the sensitivity of monocytes and macrophages to stimuli such as LPS and accelerates intracellular cascades in response to these stimuli.⁷⁰Early polarization of donor T cells so that they secrete less IFN-γ and more IL-4 can also attenuate experimental acute GVHD.⁷¹ IFN-γ may amplify GVHD by directly damaging epithelium in the GI tract and skin and inducing immnosuppression through the induction of nitric oxide.⁷² By contrast, IFN-γ may suppress GVHD by hastening the apoptosis of activated donor T cells.^69, 73. This complexity means the manipulation of IFN-γ may have diverse effects in vivo, making it a challenging target with respect to therapeutic intervention. IL-10 plays a key role in suppression of immune responses, and clinical data suggest it may regulate acute GVHD.¹⁷ TGF-β, another suppressive cytokine can suppress acute GVHD but exacerbate chronic GVHD.⁷⁴ Thus the timing and duration of the secretion of any given cytokine may determine the specific effects of that cytokine on GVHD severity.

Phase III: Cellular and Inflammatory Effector Phase

The effector phase of this process is a complex cascade of both cellular mediators such as cytotoxic T lymphocytes(CTLs) and NK cells and soluble inflammatory mediators such as TNF-α, IFN-γ, IL-1 and nitric oxide.^2, 29 These soluble and cellular mediators synergize to amplify local tissue injury and further promote inflammation and target tissue destruction.

Cellular Effectors

The cellular effectors of acute GVHD are primarily CTLs and NK cells.⁴⁹ CTLs that preferentially use the Fas/FasL pathway of target lysis and appear to predominate in GVHD liver damage (hepatocytes express large amounts of Fas) whereas GVHD CTLs that use the perforin /granzyme pathways are more important in the GI tract and skin.^2, 75 Chemokines direct the migration of donor T cells from lymphoid tissues to the target organs where they cause damage. Macrophage inflammatory protein-1alpha (MIP-1α) and other chemokines such as CCL2-5, CXCL2, CXCL9-11, CCL17 and CCL27 are over-expressed and enhance the homing of cellular effectors to target organs during experimental GVHD.⁷⁶Expression of integrins, such as α4β7 and its ligand MadCAM-1, are also important for homing of donor T cells to Peyer’s patches during intestinal GVHD.^52, 77, 78

Prevention of GVHD

Based on the evidence from animal models regarding the central role of T cells in initiating GVHD, numerous clinical studies evaluating T cell depletion (TCD) as prophylaxis for GVHD were performed in the 1980’s and 1990’s. There were three principal TCD strategies: (1) negative selection of T cells ex vivo, (2) positive selection of CD34⁺ stem cells ex vivo; and (3) anti-T cell antibodies in vivo.⁸³Most strategies showed a significant limitation in both acute and chronic GVHD.^84–88 Unfortunately, the lower incidence of severe GVHD was offset by high rates of graft failure, relapse of malignancy, infections, and Epstein-Barr virus-associated lymphoproliferative disorders. Negative selection purging strategies using various anti-T cell antibodies achieved similar long-term results regardless of the breadth of antibody specificity.^89–93 One large registry study demonstrated that purging strategies using antibodies with broad specificities produced inferior leukemia-free survival than standard immunosuppression in patients receiving unrelated donor transplants.⁹⁴ Several studies have investigated partial T cell depletion, either by eliminating specific T cell subsets (e.g., CD8⁺) or by titrating the dose of T cells present in the inoculum.^95–97 None of these approaches, however, has convincingly demonstrated an optimal strategy that improves long-term survival.

Alemtuzumab is a monoclonal antibody that binds CD52, a protein expressed on a broad spectrum of leukocytes including lymphocytes, monocytes, and dendritic cells. Its use in GVHD prophylaxis in a Phase II trial decreased the incidence of acute and chronic GVHD following reduced intensity transplant.⁹⁸ In two prospective studies, patients who received alemtuzumab rather than methotrexate showed significantly lower rates of acute and chronic GVHD,⁹⁹ but experienced more infectious complications and higher rates of relapse, so that there was no overall survival benefit. Alemtuzumab may also contribute to graft failure when used with minimal intensity conditioning regimens.¹⁰⁰

An alternative strategy to TCD attempted to induce anergy in donor T cells by ex vivo antibody blockade of co-stimulatory pathways prior to transplantation. A small study using this approach in haploidentical HCT recipients was quite encouraging, but has not yet been replicated.¹⁰¹ Thus the focus of most prevention strategies remains pharmacological manipulation of T cells after transplant.

Administration of anti-T cell antibodies in vivo as GVHD prophylaxis has also been extensively tested. The best studied drugs are anti-thymocyte globulin (ATG) or antilymphocyte globulin (ALG) preparations. These sera, which have high titers of polyclonal antibodies, are made by immunizing animals (horses or rabbits) to thymocytes or lymphocytes, respectively. A complicating factor in determining the role of these polyclonal sera in transplantation is the observation that even different brands of the same class of sera exert different biologic effects.¹⁰² However, the side effects of ATG/ALG infusions are common across different preparations and include fever, chills, headache, thrombocytopenia (from cross-reactivity to platelets), and, infrequently, anaphylaxis. In retrospective studies, rabbit ATG reduced the incidence of GVHD in related donor HSCT recipients without appearing to improve survival.^103, 104 In recipients of unrelated donor HSCT, addition of ALG to standard GVHD prophylaxis effectively prevented severe GVHD, but did not result in improved survival because of increased infections.¹⁰⁵ In a long term follow-up study, however, pretransplant ATG provided significant protection against extensive chronic GVHD and chronic lung dysfunction.¹⁰⁶

The primary pharmacologic strategy to prevent GVHD is the inhibition of the cytoplasmic enzyme, calcineurin, that is critical for in the activation of T cells. The calcineurin inhibitors, cyclosporine and tacrolimus, have similar mechanisms of action, clinical effectiveness and toxicity profiles, including hypomagnesemia, hyperkalemia, hypertension, and nephrotoxicity.^9, 107 Serious side effects include transplant-associated thrombotic microangiopathy (TAM) and neurotoxicity that can lead to premature discontinuation. Although clinically similar to thrombotic thrombocytopenic purpura, TAM does not reliably respond to therapeutic plasmapheresis, carries a high mortality rate, and removal of the offending agent does not always result in improvement.¹⁰⁸ Posterior reversible encephalopathy syndrome includes mental status changes, seizures, neurological deficits and characteristic magnetic resonance imaging findings; this syndrome has been seen in 1-2% of HCT recipients receiving and calcineurin inhibitors.¹⁰⁹ Side effects of these drugs decrease as the dose is tapered, usually two to four months after HCT.

Calcineurin inhibitors are often administered in combination with other immunosuppressants, such as methotrexate, which is given at low doses in the early post-transplant period.^9, 107 The toxicities of methotrexate (neutropenia and mucositis) have led some investigators to replace it with mycophenolate mofetil (MMF). In one prospective randomized trial, patients who received MMF as part of GVHD prophylaxis experienced significantly less severe mucositis and more rapid neutrophil engraftment than those who received methotrexate.¹¹⁰ The incidence and severity of acute GVHD was similar between the two groups, but the study closed early due to superiority of the MMF arm with respect to reduced mucositis and the speed of hematopoietic engraftment. A desire for faster neutrophil engraftment has led to the use of MMF in UCB blood transplants where graft failure is a major concern.¹¹¹ MMF is also often used after RIC regimens for similar reasons.^112, 113

Sirolimus is an immunosuppressant that is structurally similar to tacrolimus but does not inhibit calcineurin. In a small Phase II trial, it showed excellent efficacy in combination with tacrolimus;¹¹⁴ the drug damages endothelial cells, however, and it may enhance TAM that is associated with calcineurin inhibitors.¹¹⁵ The combination of tacrolimus and sirolimus is currently being compared in a large randomized multi-center trial.

RIC regimens attempt to suppress the host immune system sufficiently so that donor T cells can engraft and then ablate the lympho-hematopoietic compartment of the recipient. The term “non-myeloablative” is therefore somewhat misleading. RIC regimens produce less tissue damage and lower levels of the inflammatory cytokines that are important in the initiation of GVHD pathophysiology; this effect may explain the reduced incidence of severe GVHD following RIC compared to the full intensity conditioning used in historical controls.^98, 116 The onset of acute GVHD may be delayed after RIC until after day 100, however, and it may present simultaneously with elements of chronic GVHD (“overlap syndrome”).^116–120

Treatment of Acute GVHD

GVHD generally first develops in the second month after HCT, during continued treatment with calcineurin-based prophylaxis.^23, 121 Steroids, with their potent antilymphocyte and anti-inflammatory activity, are the gold standard for treatment of GVHD. Many centers treat mild GVHD of the skin (Grade I) with topical steroids alone, but for more severe skin GVHD and any degree of visceral GVHD involvement, high-dose systemic steroids are usually initiated. Steroid therapy results in complete remission in less than half of the patients,¹²² and more severe GVHD is less likely to respond to treatment.^123, 124 In a prospective randomized study, the addition of ATG to steroids as primary therapy did not increase the response rate.¹²⁴ In a retrospective study, the use of ATG in patients who showed early signs of steroid-resistance was beneficial,¹²² but not all studies show such benefit and ATG is not standardly used because of increased infection risks.^{106, 125, 126}.

An increasingly common treatment for GVHD is extracorporeal photopheresis (ECP). During ECP, the patient’s white blood cells are collected by apheresis, incubated with the DNA-intercalating agent, 8-methoxypsoralen, exposed to ultraviolet light (UVA), and returned to the patient. ECP is known to induce cellular apoptosis, which has strong anti-inflammatory effects in a number of systems, including prevention of rejection of solid organ grafts.¹²⁷ Animal studies show that ECP reverses acute GVHD by increasing the number of regulatory T cells.¹²⁸ A Phase II clinical study of steroid-dependent or steroid refractory GVHD showed resolution of GVHD in a large majority of patients, with 50% long-term survival in this very high risk group.¹²⁹ Randomized multi-center studies of this approach are needed to determine its place in the management of acute GVHD.

Another interesting strategy to treat GVHD is the blockade of the inflammatory cytokine TNF-α. TNF-α can activate APCs, recruit effector cells and cause direct tissue damage.¹³⁰ In animal models, TNF-α plays a central role in GVHD of the GI tract, which is central to the “cytokine storm” and plasma levels of TNFR I (a surrogate marker for TNF-α) rise in patients before the clinical manifestations of GVHD appear. ⁵¹ A recent Phase II trial of etanercept, a solubilized TNFR II, showed significant efficacy when added to systemic steroids as primary therapy for acute GVHD. Seventy percent of patients had complete resolution of all GVHD symptoms within one month, with 80% complete responses in the GI tract and the skin. The authors also showed that plasma levels of TNFR I were a significant biomarker for clinical GVHD.¹³¹

Treatment of Chronic GVHD

In contrast to acute GVHD, the pathophysiology of chronic GVHD remains poorly understood, and it is treated with a variety of immunosuppressive agents. The response of chronic GVHD to treatment is unpredictable, and mixed responses in different organs can occur in the same patient. Confounding variables such as infection and co-morbidities also make responses hard to measure. The use of corticosteroids (with or without a calcineurin inhibitor) is the standard of care, but a randomized trial of more than 300 patients with chronic GVHD found no difference between cyclosporine plus prednisone versus prednisone alone.¹³² Chronic immunosuppressants, especially those containing steroids, are highly toxic and result in infectious deaths. Many second line therapies have been studied, but none has achieved widespread acceptance. As mentioned above, ECP shows some promise, with significant response rates in high-risk patients. The best responses were observed in skin, liver, oral mucosa, eye, and lung.¹³³ This observation is particularly relevant because lung GVHD has the potential to be a particularly devastating complication necessitating lung transplant as the only therapeutic option.^134, 135

Essential Supportive Care in GVHD Patients

Meticulous supportive care is critical for patients with both acute and chronic GVHD because of the extended duration of immunosuppressive treatments and because the multiple medications required may have synergistic toxicities. Such care includes extensive infectious prophylaxis, early interventions in cases of suspected infections, and prophylaxis against non-infectious side effects of medications (See Table 3). These complications often require rapid responses to prevent serious or irreversible damage, and are best handled in close collaboration between the primary physician and the transplant specialist.

Table 3

Recommendations for Supportive Care

All patients should receive at least fluconazole as prophylaxis against fungal infections. Invasive molds, especially aspergillus, are common in patients with prolonged steroid use.¹³⁶ Prophylaxis with voriconazole or posaconazole should be considered for these patients. Usual sites of infection are the lungs, sinuses, brain, skin,¹³⁷ and serial galactomannan assays may aid in the early detection.¹³⁸ Candida can cause lesions in the lung and spleen, which may need screening with ultrasonography. Pneumocystis is another opportunistic infection that should receive cotrimoxazol (bactrim) prophylaxis.¹³⁹

Viral infections are frequent in these patients with GVHD. Cytomegalovirus causes interstitial pneumonia and gastritis. Patients who are at risk should have their blood monitored several times monthly. Techniques that directly detect virus should be performed, such as CMV PCR or pp65 antigen, and evidence of increased viral load should prompt preemptive treatment with ganciclovir or foscarnet prior to clinical manifestations of disease. Shingles is not uncommon and acyclovir prophylaxis may be beneficial.¹⁴⁰ Patients and caregivers should receive vaccinations against influenza, and treatment with neuraminidase inhibitors is recommended in the event of influenza infection.^141, 142

Patients with GVHD often have IgG₂ and IgG₄ subclass deficiencies despite normal lgG levels, making them susceptible to infections with encapsulated organisms. Treatment of severe hypogammaglobulinemia with intravenous immunoglobulin is standard in many centers,¹⁴³ but the level that triggers replacement varies considerably among transplant specialists. There is little supporting evidence for the routine use of intravenous immunoglobulin as prophylaxis¹⁴⁴ but patients should receive routine prophylaxis (penicillin or its equivalent) due to the increased risk of streptococcal sepsis.¹⁴⁵ Pneumococcal conjugate and hemophilus influenza vaccine may provide additional protection and are also recommended for all patients, including those with chronic GVHD.^{139, 146, 147} The sites of any indwelling catheters should be assessed regularly and early treatment of a suspected infection initiated. Early signs or symptoms of septic shock such as shaking chills or low blood pressure requires prompt evaluation with chest X-ray and/or CT scan, blood culture and broad spectrum antibiotics because shock may progress rapidly in these patients.

9.3.5 Aspergillus Complicating Allogeneic Transplantation

Aspergillus infections in allogeneic stem cell transplant recipients: have we made any progress?

E Jantunen, V-J Anttila and T Ruutu
BMT 2002; 30(12):925-929
http://www.nature.com/bmt/journal/v30/n12/full/1703738a.html
http://dx.doi.org:/10.1038/sj.bmt.1703738

Invasive aspergillosis (IA) is common in allogeneic SCT recipients, with an incidence of 4-10%. The majority of these infections are diagnosed several months after SCT and they are frequently associated with GVHD. The diagnosis is difficult and often delayed. Established IA is notoriously difficult to treat with a death rate of 80-90%. This review summarises recent data on this problem to assess whether there has been any progress. Effective prophylactic measures are still lacking. Severe immunosuppression is the main obstacle to the success of therapy. Recent and ongoing developments in diagnostic measures and new antifungal agents may improve treatment results to some extent, but Aspergillus infections still remain a formidable problem in allogeneic transplantation. Further studies in this field will focus on the role of various cytokines and combinations of antifungal agents.

Summary

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