Biol Blood Marrow Transplant 19 (2013) 1537e1545

Report

Proceedings from the National Cancer Institutes Second ASBMT

American Society for Blood
and Marrow Transplantation
International Workshop on the Biology, Prevention, and
Treatment of Relapse after Hematopoietic Stem Cell
Transplantation: Part I. Biology of Relapse after
Transplantation
Ronald E. Gress 1, Jeffrey S. Miller 2, Minoo Battiwalla 3, Michael R. Bishop 4,
Sergio A. Giralt 5, Nancy M. Hardy 1, *, Nicolaus Krger 6, Alan S. Wayne 7, 8,
Dan A. Landau 9, Catherine J. Wu 9
From the 1 Experimental Transplantation Immunology Branch, National Institutes of Health, National Cancer Institute, Center for Cancer Research, Bethesda, Maryland
2
Hematology, Oncology and Transplantation, University of Minnesota Cancer Center, Minneapolis, Minnesota
3
Hematology Branch, National Heart, Lung and Blood Institute, Hematology Branch, Bethesda, Maryland
4
Section of Hematology/Oncology, University of Chicago, Chicago, Illinois
5
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York
6
Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
7
Pediatric Oncology Branch, National Institutes of Health, National Cancer Institute, Center for Cancer Research, Bethesda, Maryland
8
Current: Childrens Center for Cancer and Blood Diseases, Childrens Hospital Los Angeles, Keck School of Medicine, University of
Southern California, Los Angeles, California
9
Cancer Vaccine Center and Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts

Article history: a b s t r a c t
Received 24 August 2013 In the National Cancer Institutes Second Workshop on the Biology, Prevention, and Treatment of Relapse
Accepted 30 August 2013 after Hematopoietic Stem Cell Transplantation, the Scientic/Educational Session on the Biology of Relapse
discussed recent advances in understanding some of the host-, disease-, and transplantation-related
Key Words: contributions to relapse, emphasizing concepts with potential therapeutic implications. Relapse after
Stem cell transplantation hematopoietic stem cell transplantation (HSCT) represents tumor escape, from the cytotoxic effects of the
Relapse conditioning regimen and from immunologic control mediated by reconstituted lymphocyte populations.
Allogeneic
Factors inuencing the biology of the therapeutic graft-versus-malignancy (GVM) effectdand rela-
Prevention
psedinclude conditioning regimen effects on lymphocyte populations and homeostasis, immunologic niches,
Biology
Treatment and the tumor microenvironment; reconstitution of lymphocyte populations and establishment of functional
immune competence; and genetic heterogeneity within the malignancy dening potential for clonal escape.
Recent developments in T cell and natural killer cell homeostasis and reconstitution are reviewed, with
implications for prevention and treatment of relapse, as is the application of modern genome sequencing to
dening the biologic basis of GVM, clonal escape, and relapse after HSCT.
Published by Elsevier Inc. on behalf of American Society for Blood and Marrow Transplantation.

INTRODUCTION processes that depend on recovery of thymic function and

Recovery of immunologic competence contributes to the
therapeutic efcacy of hematopoietic stem cell trans-
plantation (HSCT). T lymphocytes and natural killer (NK)
cells have demonstrated antitumor potential in the alloge-
neic transplant (AlloSCT) setting and may have therapeutic
benet after autologous transplantation (AHSCT) as well.
Successful transplant outcomes require the recovery of
functional immune competence, including reconstitution of
immune effectors from populations transferred in the graft,
through peripheral expansion of mature T cells and deriva-
tion of new lymphocyte populations from progenitors, and
the establishment of antigen-presenting cell and regulatory
cell populations. Complete immune reconstitution includes
recovery of repertoire and control of autoreactivity,
Financial disclosure: See Acknowledgments on page 1544.
* Correspondence and reprint requests: Nancy M. Hardy, MD, Experi-
mental Transplantation and Immunology Branch, National Cancer Institute/
Center for Cancer Research, 10 Center Drive, Hateld Clinical Research
Center, Room 3E-3330, Bethesda, MD 20892.
E-mail address: hardyn@mail.nih.gov (N.M. Hardy).
1083-8791/$ e see front matter Published by Elsevier Inc. on behalf of American Society for Blood and Marrow Transplantation.
http://dx.doi.org/10.1016/j.bbmt.2013.08.010
development of nave and regulatory T cell (Treg) pop-
ulations. Peripheral recovery from lymphopenia is marked
by vulnerability to dysregulated immune responses and
immune incompetence. It also represents a period of ther-
apeutic opportunity, with potential to direct antigen-driven
expansion toward tumor targets.
LYMPHOCYTE HOMEOSTASIS: IMPLICATIONS FOR THE
PREVENTION AND TREATMENT OF RELAPSE
Loss of Lymphocyte Populations with Conditioning
A generalized loss of lymphocyte populations is seen with
most chemotherapy regimens; such loss can be profound
after even a single cycle of conventional outpatient therapy.
Myeloablative conditioning regimens are highly lymphoa-
blative, leaving only small residual lymphocyte populations.
However, after reduced-intensity conditioning, incomplete
host lymphoid depletion provides for potential expansion of
residual host populations along with donor T cell expansion,
adding another layer of complexity to the biology of T cell
reconstitution after AlloSCT.
1538 R.E. Gress et al. / Biol Blood Marrow Transplant 19 (2013) 1537e1545
Limitations to Lymphocyte Recovery after HSCT
Conditioning
Some functional lymphocyte and antigen-presenting cell
populations are regenerated from graft-derived progenitors
within a few months, including monocytes, dendritic cells,
and NK cells [1,2]. In the absence of B-lymphocyte directed
therapy, circulating B cell counts recover within 3 months,
but functional decits persist unless T cell subset recovery
permits the CD4 T cell help necessary for memory B cell
development. Memory and effector T cells in the graft,
particularly CD8 subsets, can expand in response to anti-
genic stimulation [3], but regeneration of T cells with
a diverse repertoire of T cell receptors (TCR) requires
renewed thymopoiesis. The potential for thymic recovery
after HSCT declines sharply with age [4] and may be
hindered by tissue damagedfrom the conditioning regimen
and, after AlloSCT, from acute graft-versus-host disease
(GVHD). Resultant thymic epithelial cell damage interferes
with proper clonal deletion of autoreactive T cells and
reduces quantitative T cell production, including the gener-
ation of Tregs. Resultant thymic epithelial cell damage may
lay the foundation for later development of the
autoimmune-like dysfunction of chronic GVHD.
General Pathways for Regeneration of T Cell Subsets
Murine studies identied 2 pathways of T cell regeneration:
thymic-dependent maturation of new T cells from marrow
progenitors and thymic-independent expansion of mature
peripheral T cells [5-9]. Through the latter, T cell numbers are
maintained by a balance between their cytokine consumption
and levels of constitutively expressed homeostatic cytokines
(IL-7 and IL-15) that support their maturation, proliferative
expansion, and survival. Homeostatic peripheral expansion in
lymphopenic hosts can be strongly skewed by rapid, antigen-
driven proliferation. Translational studies in children, which
validated CD45 isoform-dened CD4 T cell subsets, demon-
strated recovery of total and nave (CD45CD45RO) periph-
eral CD4 T cells as early as 6 months after severe
chemotherapy-induced lymphopenia and established thymic-
dependent T cell production as primarily responsible for this
repopulation in young patients [6]. In contrast, in adults, few
nave peripheral CD4 Tcells are generated during the rst year
after chemotherapy, whereas circulating CD4 T cells with
a memory phenotype (CD45RO) recover to nearly pretreat-
ment levels (w83%) within 3 months through thymus-
independent peripheral expansion [10].
Longitudinal study of T cell recovery in adults after mye-
loablative conditioning and AHSCT for breast cancer demon-
strated the inuence of age-associated thymic renewal on
CD4 T cell recovery. In patients in whom renewal of thy-
mopoiesis was observeddas assessed by nave cell pheno-
type (CD45RA), T cell receptor excision circle frequency,
thymic size, and rediversication of TCR repertoirednorm-
alization of circulating CD4 T cell counts was noted by the
end of the second year and permitted reestablishment of
central memory populations. In those who did not reactivate
thymopoiesis, CD4 T cell counts remained below normal
even up to 5 years after AHSCT [4]. Recovery of CD8 T cells in
the same patient cohort followed markedly different repo-
pulation kinetics, with 3 distinct patterns observed (Figure 1).
In more than half of the patients, CD28CD8 T cell expansion
yielded a massive early spike in total peripheral CD8 T cell
counts and constituted the dominant population in persis-
tently elevated CD8 T cells for 2 years after AHSCT. The
circulating CD8 T cell population demonstrated limited TCR
repertoire diversity, with a high proportion of oligoclonal,
expanded CD28 cells by TCR spectratyping that was strongly
associated with a history of cytomegalovirus (CMV) infection,
suggesting viral antigen-driven peripheral expansion. In
younger adult patients, CD8 T cell recovery bore resem-
blance to that of CD4 T cells: phenotypically nave CD8 T
cells gradually predominated the circulating CD8 T cell pool
during the second year, generating a population with
a broadly diverse TCR repertoire and high T cell receptor
excision circle frequencies resulting from thymic reactivation.
A subset of younger patients also demonstrated the early,
oligoclonal expansion of CD8 T cells, suggesting that
peripheral and thymic pathways were independent modula-
tors of recovery. A third pattern of CD8 T cell repopulation
was characterized by the absence of both the early expansion
of effectors and the later recovery of nave CD8 T cells; these
patients experienced prolonged, severe decits in circulating
T cell numbers and in reconstitution of TCR repertoire diver-
sity in CD4 as well as CD8 T cell subsets (Figure 1). This third
pattern exhibits the limits of thymus-independent homeo-
static expansion in reconstituting functional immune
competence in middle-aged adults.
The regeneration of Tregs is less well characterized than
for the conventional T cell subsets described. It appears that
Treg repopulation also can derive from thymus-dependent
and peripheral expansion pathways in the setting of lym-
phopenia [11] and that thymic renewal may yield Treg pop-
ulations capable of modulating chronic GVHD [12]. Exogenous
IL-2 administration increases the number of circulating Tregs
through both pathways [13], with therapeutic potential for
prevention and treatment of chronic GVHD [14].
Implications for Prevention and Treatment of Relapse
A better understanding of physiologic regulation of
thymic function is needed to identify clinical strategies to
optimize the thymus-dependent pathway, particularly in
adults. Insulin-like growth factor 1, androgen blockade,
and keratinocyte growth factor are candidate regulators
amenable to clinical approaches to enhance recovery of
thymopoiesis and of functional T cell immunity. Further,
clinical availability of primary homeostatic cytokines (ie, IL-7
and IL-15) creates the possibility of enhancing the peripheral
expansion pathway and tumor antigenedriven expansion
after HSCT and/or in conjunction with tumor-specic
immunotherapies. Similarly, our developing understanding
of the biology of Treg homeostasis may yield opportunity for
relapse prevention through GVHD modulation (eg, IL-2). As
functionally distinct T cell subsets continue to be identied,
some (eg, memory stem T cells [15]) may be important
determinants of functional immune reconstitution after
recovery from lymphopenia. The biology of emerging T cell
subsets will need to be integrated into our understanding of
T cell homeostasis and immune reconstitution.
Recent advances in cellular engineering create potentially
powerful tools for generating cancer-specic immune
responses. The development of genetically modied effector
T cells now permits deliberate skewing of the T cell repertoire
toward antitumor effector populations. Chimeric antigen
receptors redirect T cell specicity and function through
combining a specic antibodys antigen-recognition domain
with a T cell signaling domain yielding a receptor with high
specicity and independence from TCReMHC restriction
[16]. The biology of homeostatic antigen-driven peripheral
expansion may be exploited to favor proliferation of adop-
tively transferred cells; timing administration to coincide
 R.E. Gress et al. / Biol Blood Marrow Transplant 19 (2013) 1537e1545 1539

Figure 1. Diagrammatic representation of the timeline of immune reconstitution in a 40-year-old CMV-seropositive adult. After cytoreductive therapy, the peripheral
CD4 and CD8 T cell populations are severely depleted. The thymus is reduced to a small remnant (shown at 1.5 months post-transplant). CD4 and CD8 T cells
immediately undergo a marked expansion in response to homeostatic cytokines and endogenous antigens, generating a population that is mainly composed of
memory (green) and effector (orange) T cells, with few nave (blue) or T cell receptor excision circle (TREC)ebearing cells (also represented at 1.5 months post-
transplant). The CD4/CD8 ratio becomes inverted by the more rapid expansion of CD8 T cells, demonstrated by the peak of (orange) effector CD8 cells as
compared with the smaller (green) peak of CD4 cells at 1.5 months. TCR repertoire diversity that has been lost by lymphodepletion is further skewed by oligoclonal
expansion of the limited number of remaining cells. The expanded population of CD8 T cells persists and may continue to dominate the CD8 TCR repertoire as shown
in subsequent months by the large effector (orange) CD8 population. Renewed thymopoiesis begins within the rst 6 months, but the full contribution of nave, TREC-
bearing T cells with a diverse TCR repertoire may require 1 to 2 years to be evident. (Reprinted from Williams KM, Hakim FT, Gress RE. T cell immune reconstitution
following lymphodepletion. Semin Immunol 2007;19:318-330, 2007. Copyright (2007), with permission from Elsevier.)

with therapy-induced lymphopenia and/or coadministration
of exogenous homeostatic cytokines are potentially syner-
gistic immunomodulatory strategies to support their survival
and expansion in vivo. Interruption of counter-regulatory
mechanisms may improve antitumor responses as well,
with Treg depletion and checkpoint inhibition (eg, pro-
grammed cell death protein 1 (PD-1) PD-1 [17]) as examples
of promising strategies.
AHSCT-associated lymphopenia also provides an ad-
vantageous milieu for homeostatic expansion and an
opportunity to support antigen-directed immunotherapy
by vaccines or other immunomodulatory approaches.
Remarkable recent progress in using such strategies was
discussed in Sessions II and III. Perhaps for the rst time in
the history of HSCT, we have arrived at an intersection of
knowledge and technology that may permit removal of the
1540 R.E. Gress et al. / Biol Blood Marrow Transplant 19 (2013) 1537e1545

fundamental barriers that have limited this eld from its
inception.
NK CELL BIOLOGY AND THERAPEUTIC POTENTIAL
Our understanding of the heterogeneous and complex NK
cell cytotoxic lymphocyte population evolved from initial
murine transplant studies showing lymphocytes with MHC-
unrestricted cytotoxic antitumor activity in vitro; current
biology incorporates the subsequent identication of
a distinct NK cell lymphocyte population, NK inhibitory
receptors (the LY49 receptor family in mice and inhibitory
killer immunoglobulin-like receptors [KIR] and NKG2A/CD94
heterodimer in humans), and, more recently, MHC-inde-
pendent activating receptors in both mouse and humans.
Ontogeny, homeostatic regulation, and function of NK cells
remain areas of active investigation, but it appears they
derive from common lymphoid progenitors in the bone
marrow and undergo IL-15edependent development in
secondary lymphoid organs, with terminal differentiation
and cytotoxic function acquired in the periphery. Their
function is complex, apparently playing important roles in
both tolerance (ie, MHC-disparate fetal tissue, with an
abundance of CD56-bright, immature NK cells predominat-
ing the uterine leukocyte population) and surveillance, for
example, killing of tumor and virally infected cells that evade
T cells via reduced MHC cell-surface expression (with
a CD56-dim, terminally differentiated population). Such
complexity was also rst observed in murine transplant
models that demonstrated NK cell effects on GVHD modu-
lation and graft-versus-malignancy and in inspired human
studies that demonstrated NK cell graft-versus-malignancy
activity in acute myelogenous leukemia [18-20]; Dr. Hsu
discussed the implications of NK cell immunogenetics for
relapse prevention in Session II.
Inhibitory KIR are the main human family of MHC Class I
binding receptors that recognize HLA-Bw4 (present on some
HLA-B and HLA-A alleles) and HLA-C group ligands. KIR are
complex because they can also mediate activation based on
association with adaptor proteins that stimulate function.
Although the KIR family has dominated the clinical literature,
the net result of whether or not an NK cell kills its target is
determined by a large number of activating and inhibitory
interactions independent of MHC binding. Thus, allor-
eactivity is determined by a lack of inhibition through Class I
recognizing receptors and a positive balance of activating
signals including but not limited to activating KIR, CD16
(FcRgIII), natural cytotoxicity receptors (NKp30, 44, 46),
DNAM-1, LFA-1, NKG2C, NKG2D, and CD244 (2B4) (Figure 2).
The mechanism by which NK cells acquire self-tolerance
and alloreactivity has been referred to as NK cell education
or licensing [21]. Simply stated, this is the process by which
NK cells acquire function through interactions regulated by
Class I recognizing inhibitory receptors (inhibitory KIR and
signaling through NKG2A/CD94 heterodimers that bind HLA-
E). In addition to licensing, NK cells can acquire function
through several activating mechanisms, including cytokine
stimulation and inammation induced by viral infection.
Adding to these complexities, specic conditions for acti-
vating components of the KIR family remain largely unde-
ned; for example, whereas KIR2DS1 recognizes HLA-C2 at
low afnity [22], the ligand for other activating KIR is
unknown.
Insights from the KIR-homologous murine Ly49 system
may prove informative. The activating receptor Ly49H
recognizes the murine CMV glycoprotein m157, providing
proof-of-principle that activating receptors may recognize
viral proteins [23]. Activation through Ly49H may be effec-
tive on NK cells without classic licensing mechanisms.

Figure 2. NK cells express a number of inhibitory and activating receptors that determine function. Some of these interactions are denitively established with
known signaling pathways, whereas others are less clear. Most receptors interact with cellular targets, but CD16 delivers a potent signal by binding the Fc portion of
immunoglobulin-coated targets. The ability of NK cells to kill a target is determined by the net balance of these inhibitory, activating, antibody-dependent, and
adhesion interactions. (Reprinted from Murphy WJ, Parham P, Miller JS. NK cellsdfrom bench to clinic. Biol Blood Marrow Transplant 2012;18:S2-S7. Copyright
(2012), with permission from Elsevier.)
 R.E. Gress et al. / Biol Blood Marrow Transplant 19 (2013) 1537e1545
Ly49H NK cells expand and then contract after viral control,
yet these cells can persist in the recipient 3 months after
infection, suggesting existence of NK cell memory, further
supported by their heightened responsiveness (such as IFN-g
production) upon secondary challenge [24].
Functional Activity of NK Cells Reconstituting Early after
Transplantation
After AlloSCT, NK cells are the rst lymphocyte population
to recover, with donor cells predominant. NK cells are
attractive to exploit in the setting of HSCT because their early
recovery occurs when the adaptive immune system is
particularly weak. However, the repopulating NK cells that
are circulating early after AlloSCT are phenotypically and
functionally different from the highly functional, FcRgIII-
bearing CD56dim NK cells found in peripheral blood from
healthy donors. These early NK cells may be developmentally
immature, with diminished KIR expression and higher
expression of NKG2A [25] and CD56] CD56bright NK cells are
a normally small population in the periphery (usually found
in secondary lymphoid tissues and the pregnant uterus);
although they respond to IL-12 and IL-18 with high IFN-g
production, they lack cytotoxic effector capabilities. NK cell
responses to targets are defective, especially for target-
induced cytokine production [26]. These responses
normalize over the rst year after HSCT, coinciding with T
cell recovery and suggesting that T cells may play a role in the
acquisition of NK cell maturation and cytotoxic function,
possibly through the cytokines they release.
Endogenous Pathways and Pitfalls to Enhanced NK Cell
Function
Based on murine studies with CMV and Ly49H NK cells,
human studies suggest that the C-type lectin-like receptor
NKG2C and the KIR family may be involved in viral infection.
We studied NK cell function after human CMV reactivation
following umbilical cord blood AlloSCT [27]. In this setting,
recipient CMV reactivation would recapitulate a primary
immune response from the CMV-nave donor NK cells.
Patient blood samples were collected at the time of reac-
tivation and at 2, 4, and 8 weeks after initiating antiviral
therapy. NKG2C NK cells increased after detection of virus
in the blood, peaking 4 weeks after reactivation. NK cells
expressing NKG2C produced signicantly more IFN-g after
exposure to a Class I- target than NK cells lacking NKG2C.
Four weeks after reactivation, NK cells coexpressing NKG2C
and KIR produced signicantly more IFN-g than NK cells
expressing KIR but not NKG2C. In the context of education of
KIR NK cells through their MHC ligands, only NKG2C NK
cells with self-KIR (ie, NK cells that express KIR where the
cognate ligand is expressed in the recipient) made IFN-g; in
contrast, NKG2C nonself KIR NK cells were hypores-
ponsive. Remarkably, an expanded mature, highly functional
NKG2CKIR NK cell population persisted throughout the
rst year after transplant. These ndings support the
emerging concepts of NK cell memory in humans and that
NK cell responses to CMV reactivation may promote more
generalized recovery of NK cell maturation and functional
competence that could be important for protecting against
cancer relapse and/or viral infections.
Trials exploring adoptive transfer of autologous activated
NK cells have not demonstrated efcacy, presumably
because of inhibition through Class I receptor signaling [28].
Reports of the benet of allogeneic NK cells after AlloSCT
with donors expressing nonrecipient (ie, nonself) inhibitory
1541
KIR led to exploration of allogeneic NK cell adoptive transfer.
To overcome barriers of allogeneic immunity and achieve
in vivo NK cell expansion, success was dependent on a potent
lymphodepleting regimen to create space, increase en-
dogenous cytokines, and eliminate immune rejection of
adoptively transferred cells. In 2005, we identied the
requirement for lymphodepleting chemotherapy to promote
in vivo expansion of NK cells after adoptive transfer of hap-
loidentical related donor NK cells in a nontransplant setting
[29]. NK cells were infused with IL-2 administration for the
2-week interval after adoptive transfer. Chimerism studies
established unequivocally that donor-derived haploidentical
NK cells could persist and expand in vivo in some patients;
antitumor responses seemed to correlate with in vivo NK cell
expansion.
In some settings, IL-2 administration may result in Treg
expansion and contraction of the immune response [30,31].
In an attempt to further overcome these barriers, some
investigations have been exploring increased lymphodeple-
tion by adding total body irradiation, approaching myeloa-
blative intensity conditioning. In this setting, HSCT may be
warranted to avoid prolonged neutropenia. Clearly, safer
strategies are needed to maximize the effector-to-target ratio
in vivo. IL-15 administration has recently shown promise in
primates; it is being produced by the National Cancer Insti-
tute and clinical trials are in progress. The main advantage of
IL-15 is that it does not stimulate CD25hi (IL-2Ra) Treg
expansion. An alternative approach is ex vivo expansion of
NK cells, which could permit infusion of a greater cell dose
and, potentially, reduced dependence on potent, toxic
conditioning for lymphodepletion.
Future Directions in Optimizing NK Cell Therapeutics
Our understanding of NK celletarget interactions requires
more detailed exploration of the potency of specic NK
molecules and their ligands on specic target cells (including
malignant stem cells), the stages of NK maturation, and the
effect of the recipient milieu on NK cell maturation, survival,
and clonal selection. Strategies to overcome tumor-induced
immunosuppression and allogeneic rejection of adoptively
transferred cells will likely be needed. Targeting NK cells to
increase their specicity may also enhance efcacy. We have
recently been exploring the potent signal delivered through
CD16] Bi- or trispecic killer cell engagers (BiKEs and
TriKEs)dcomprised of 1 or 2 single-chain Fv for a target-
specic antigen and a single-chain Fv capable of triggering
CD16dseem to be especially potent for this purpose [32].
Further studies will determine the most favorable context for
NK cell therapy, how best to promote in vivo survival needed
for clinical response, and how to optimize their specicity in
a minimal residual disease setting after transplantation.
INTRATUMORAL HETEROGENEITY AND CLONAL
EVOLUTION: DETECTION, IMPACT, AND THERAPEUTIC
CHALLENGES
From a clinicians perspective, relapsed hematopoietic
malignant disease is almost uniformly more resistant to
therapy than the primary disease. Thus, at the level of the
most crucial phenotypic featuredtherapy responsive-
nessdrelapsed disease is clearly not the same disease as at
diagnosis. Given the genetic underpinnings of cancer,
phenotypic evolution must, at its core, have an underlying
genotypic evolution. Originfally theorized by Nowell [33],
genetic instability is expected to lead to heterogeneity as the
neoplasm progresses, resulting in diverse and genetically
1542 R.E. Gress et al. / Biol Blood Marrow Transplant 19 (2013) 1537e1545

Figure 3. Potential treatment effects on clonal heterogeneity and disease behavior. Interclonal equilibrium can remain remarkably stable for years (top). However,
when an aggressive minor clone arises (bottom), clonal evolution begins and can be potentially accelerated by therapy. This may be due to differential resistance of
subpopulations to treatment. Additionally, a mass extinction event, such as chemotherapy, may accelerate evolution by removing the strong incumbent and allowing
the tter rising subclone to repopulate the compartment more efciently, as sometimes seen with bottlenecks in population genetics. (Adapted from Landau DA, et al.
Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell 2013;152:714-726.)
 R.E. Gress et al. / Biol Blood Marrow Transplant 19 (2013) 1537e1545
distinct subpopulations within the neoplasm. Furthermore,
a Darwinian selection process can then promote the
outgrowth of tter subclones, thus constantly reshaping the
overall tness of the population. Evidence supporting this
paradigm has accumulated over the past 4 decades with
increasing technologic sophistication, including cellular,
cytogenetic, and Sanger sequencing (rst-generation
sequencing) based methods.
The advent of next-generation sequencing has now
enabled the unprecedented ability to systematically discover
key genetic alterations that underlie cancer. This technology
has been applied across various blood malignancies and has
revealed the high degree of molecular variation that exists
between individuals with the same hematologic malignancy
(ie, intertumoral genetic heterogeneity, as reported by Wang
et al. [34], for example). However, because next-generation
sequencing has the potential for sensitive genome-wide
detection of mutations harbored in only a fraction of the
total cell population (as small as .02% [35]), an even more
startling discovery has been the detection of pervasive
genetic heterogeneity within malignant cells of the same
individual (ie, intratumoral heterogeneity).
In analyses using whole genome or whole exome
sequencing, clusters of genetic alterations of similar allelic
frequency can mark and allow the quantication of distinct
subpopulations harboring them. Various investigators have
used this strategy to characterize the number and dynamics
of subpopulations within diverse blood malignancies
[36,37]. These studies have all demonstrated a high degree
of clonal heterogeneity and striking changes in the genetic
makeup of the disease on relapse. One important insight
arising from these studies is that a branching rather than
linear pattern of evolution is more commonly observed.
This pattern implies that genetic evolution results from
complex tness equilibrium of highly diverse populations
rather than a clear succession of selective sweeps. This
nding is all the more surprising in hematologic malig-
nancies where the mixing of different cellular compart-
ments is supposedly higher than in solid malignancies and
the mutation rate is lower (Gad Getz, personal communi-
cation, Broad Institute of Massachusetts Institute of Tech-
nology and Harvard, 2012), both traits that in theory should
have favored a more linear pattern.
Another insight from these studies is that the coexisting
subclonal populations also harbor driver lesions that are ex-
pected to provide tness advantage [38], suggesting that the
growth of subclones is limited by mutual competition, so-
called clonal interference. Thus, the evolutionary dynamics
of tumor subpopulations likely result in the coexistence of
clonal variants. Future studies are anticipated to more fully
address the level of heterogeneity at the microscopic pop-
ulation level (eg, by deeper sequencing or by single-cell
genomic sequencing) and to what degree those subpopula-
tions interact [39]. Clonal dynamics are illustrated in Figure 3.
A further layer of complexity to understanding clonal
heterogeneity in hematologic malignancies involves the
concept of cancer stem cells, in which genetic hierarchy is
inuenced by the hierarchy of tumorigenic capacity. For
example, in chronic myelogenous leukemia, the sensitivity to
imatinib is determined not only by genetic alterations to
BCR-ABL but also by decreased sensitivity of leukemic
progenitor cells to this targeted therapy [40]. Therefore,
differentiation states that underlie phenotypic heterogeneity
contribute to the overall clonal diversity and have been
linked to crucial clinical outcomes [41].
1543
Finally, it should be noted that although not every genetic
alteration translates into a phenotypic difference, the accu-
mulation of large numbers of passenger mutations could
have more subtle phenotypic implications. Silent mutations
might potentially increase the phenotypic plasticity of cancer
cells by altering transcriptional networks or epigenetic
landscapes. They may also act to enhance or suppress the
combined effects of multiple altered genetic elements, which
then alter the evolutionary tness of the subclone harboring
them.
The understanding that malignant disease is composed of
a myriad of diverse subpopulations that have the ability to
transform and adapt poses a challenge to our traditional
diagnostic schemes. A disease can no longer be dened as
a single entity containing a uniform set of genetic abnor-
malities that is prognostically useful. Of note, reports suggest
that relapse clones could be traced back to minor clones
present at initial diagnosis [36,42], suggesting that the
degree of genetic heterogeneity of a tumor is likely to be an
important determinant of therapeutic outcome [43].
This concept of clonal heterogeneity also raises related
and important questions regarding negative consequences of
cancer therapies on clonal dynamics and tumor behavior
(Figure 2). In a familiar paradigm, cancer therapy may be
actively selective for resistant clones; examples for this are
numerous, including MSH6 mismatch repair gene mutations
in recurrent glioblastoma multiforme after treatment with
temozolomide [41] and the T315I BCR-ABL mutations in
chronic myelogenous leukemia [44]. An alternative proc-
essd entirely independent of differential tness in the
context of therapydmay also contribute to the emergence of
continuously more aggressive, high-tness subclones. In this
setting, massive cell kill after effective cancer therapy, acting
as a classic evolutionary restriction point, would reset
interclonal dynamics [45]. Thus, when high-tness sub-
clones already exist within a tumor population, treatment
could favor subsequent repopulation of the tumor niche with
a more aggressive subclone, leading to a worse clinical
course and, perhaps, ultimately shorter survival than had no
treatment been given [46].
Understanding evolutionary dynamics can spark the
development of novel therapeutic paradigms. Much atten-
tion has been paid to the effectiveness of targeted therapies,
but the consistent observations of clonal evolution provide
a cautionary note about their ability to generate enduring
cancer control. An alternative approach might be to try to
maintain interclonal equilibrium at the expense of maxi-
mizing cell kill [47]. In theory, preservation of sensitive
clones could, in turn, facilitate continued suppression of
therapy-resistant clones in a competitive manner. This
understanding also supports the further renement of
existing therapies, such as immune-based approaches; in
essence, this amounts to pitting 1 complex adaptive process
against another. Here, in the case that immunity is generated
against multiple tumor-specic epitopes (eg, by vaccination
or adoptive cellular therapy), tumor control and eradication
is achieved through multipronged targeting of subclonal as
well as clonal populations. This concept helps us understand
why AlloSCT, which relies on mounting donor-derived
immunity to eliminate leukemia cells, can be curative even
in patients with clonally advanced disease [48].
CONCLUSIONS
It is apparent from these 3 aspects of the biology
of relapse after HSCT that with the impressive strides
1544 R.E. Gress et al. / Biol Blood Marrow Transplant 19 (2013) 1537e1545
made, there are many more questions than answers. A
key objective met at the National Cancer Institutes Second
Workshop on the Biology, Prevention, and Treatment of
Relapse after Hematopoietic Stem Cell Transplantation was
the establishment of a consortium of transplant investigators
who are keen to work together to further understand the
biology of relapse. The Biology Protocol Development Team
made substantial progress on the development of a multi-
institutional protocol to collect, store, and allocate the crit-
ical tissue samples necessary to study the basic mechanisms
of relapse after AlloSCT. In no other area of post-transplant
relapse is the need greater for this collective effort than it
is in the biology, in which informative research requires
relapse tissue specimens across multiple transplant plat-
forms and complex clinical outcomes. Workshop partici-
pants strongly endorsed the Biology Protocol Development
Teams recommendation that the highest priority would be
to establish a protocol for collection and banking of tumor
and blood specimens at diagnosis of relapse after AlloSCT,
along with a procedure for prioritizing research questions
and sample allocation.
ACKNOWLEDGMENT
Financial disclosure: Supported in part by the Intramural
Research Programs of the National Institutes of Health: the
National Cancer Institute/Center for Cancer Research and the
National Heart, Lung and Blood Institute.
Authorship statement: R.E.G., J.S.M., and C.J.W. contributed
equally to this work.
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