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INTERNATIONAL REVIEW OF CELL AND
MOLECULAR BIOLOGY
Series Editors
GEOFFREY H. BOURNE 1949–1988
JAMES F. DANIELLI 1949–1984
KWANG W. JEON 1967–2016
MARTIN FRIEDLANDER 1984–1992
JONATHAN JARVIK 1993–1995
LORENZO GALLUZZI 2016–
Editorial Advisory Board

KEITH BURRIDGE CARLOS LOPEZ-OTIN
AARON CIECHANOVER WALLACE MARSHALL
SANDRA DEMARIA SHIGEKAZU NAGATA
SILVIA FINNEMANN MOSHE OREN
KWANG JEON ANNE SIMONSEN
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First edition 2017
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CONTRIBUTORS
R. Blanco
Centro Nacional de Biotecnologı́a/CSIC, Madrid, Spain
E. Bremer
Department of Hematology, Section Immunohematology, University of Groningen,
University Medical Center Groningen (UMCG), Groningen, The Netherlands; University
of Exeter Medical School, Exeter, UK
C. Brenner
INSERM UMR-S 1180-LabEx LERMIT, Universite Paris-Sud, Universite Paris-Saclay,
Châtenay Malabry, France
R. Caballero
GEICAM (Spanish Breast Cancer Research Group), Madrid, Spain
L. Carmona-Rodrı́guez
F.H. Carrasco
M. Chiesa
G. Choi
University Medical Center Groningen (UMCG), Groningen, The Netherlands
M. de Bruyn
Department of Obstetrics & Gynecology, University of Groningen, University Medical
Center Groningen (UMCG), Groningen, The Netherlands
L. de la Cruz-Merino
Hospital Universitario Virgen Macarena, Seville, Spain
J. Espada
Experimental Dermatology and Skin Biology Group, Ramón y Cajal Institute for Health
Research (IRYCIS), Ramón y Cajal University Hospital, Colmenar Viejo Rd. Km 9,100,
28034 Madrid, Spain; Bionanotechnology Lab, Universidad Bernardo O´Higgins, General
Gana 1780, 8370854 Santiago, Chile
C. Figueiredo-Pereira
CEDOC, NOVA Medical School, Faculdade de Ciências Medicas, Universidade Nova de
Lisboa, Lisboa, Portugal
R. Goswami
Institute of Life Sciences, Ahmedabad University, Ahmedabad, India
ix
x Contributors
D. Hendriks
Department of Surgery, Translational Surgical Oncology, University of Groningen,
M.H. Kaplan
Wells Center for Pediatric Research, Indiana University, Indianapolis, IN, United States
R.A. Lacalle
Q. Lu
Program in Molecular and Integrative Physiological Sciences, Harvard T.H. Chan School of
Public Health, Boston, MA, United States
S. Mañes
A. Martı́n-Leal
J. Martı́n-Perez
Department of Cancer Biology, Instituto de Investigaciones Biomedicas “Alberto Sols”
CSIC-UAM, Madrid, Spain
E. Mira
R. Oh
C. Oudot
N. Palazón
Hospital Universitario Virgen Macarena, Seville, Spain
R. Panganiban
H.-R. Park
F. Rojo
Fundación Jimenez Dı́az, Madrid, Spain
V. Sánchez-Margalet
Medical School, Hospital Universitario Virgen Macarena, Seville, Spain
H.L.A. Vieira
CEDOC, NOVA Medical School, Faculdade de Ciências Medicas, Universidade Nova de
Lisboa, Lisboa; Instituto de Biologia Experimental e Tecnológica (iBET), Oeiras, Portugal
Contributors xi
P. Wagner
Z. Wang
V.R. Wiersma
CHAPTER ONE
Breast Cancer Immunology

and Immunotherapy: Current
Status and Future Perspectives
L. de la Cruz-Merino*,1, M. Chiesa†, R. Caballero†, F. Rojo{, N. Palazón*,
F.H. Carrasco†, V. Sánchez-Margalet§
*Hospital Universitario Virgen Macarena, Seville, Spain
†
{
Fundación Jimenez Dı́az, Madrid, Spain
§
Medical School, Hospital Universitario Virgen Macarena, Seville, Spain
1
Corresponding author: e-mail address: lcruz-ibis@us.es
Contents
1. Introduction 3
2. Mechanisms of the Immune Response Inhibition in BC: Role of MDSCs and
Regulatory T Cells 5
3. Immune System in the Therapeutic Response in BC. The Immunogenic
Cell Death 9
4. T-Infiltrating Lymphocytes Molecular and Genomic Characterization as a
Strategic Tool to Better Understand BC Behavior 11
5. Neoantigen Prediction: Genomic Tools for Personalized Immunotherapy 22
6. Immunotherapeutic Approaches in BC. Clinical Trials With Immune Checkpoint
Inhibitors 25
6.1 Early Results in Clinical Trials With Immune Checkpoints as Single Therapy 27
6.2 Early Results in Clinical Trials With Immune Checkpoints in the Context
of Combinations 30
6.3 Ongoing and Planned Clinical Trials With Immune Checkpoints Inhibitors
in BC 31
7. Conclusions 36
Acknowledgments 37
References 37
Abstract
Cancer immunology has gained renewed interest in the past few years due to emerging
findings on mechanisms involved in tumoral immune evasion. Indisputably, immune
edition is currently considered a critical hallmark of cancer. Basic research has revealed
TICA (Spanish Group for

On behalf of GEICAM (Spanish Breast Cancer Research Group) and GE
Cancer Immuno-Biotherapy).
International Review of Cell and Molecular Biology, Volume 331 # 2017 Elsevier Inc. 1
ISSN 1937-6448 All rights reserved.
http://dx.doi.org/10.1016/bs.ircmb.2016.09.008
2 L. de la Cruz-Merino et al.
new targets which can be modulated in the clinical setting with new compounds and
strategies. As recent evidence confirms, breast cancer (BC) is a complex and heteroge-
neous disease in which host immune responses play a substantial role. T-infiltrating
lymphocytes measurement is suggested as a powerful new tool necessary to predict
early BC evolution, especially in HER2-positive and triple negative subtypes. However,
T-infiltrating lymphocytes, genomic platforms, and many other biomarkers in tissue
and peripheral blood (e.g., regulatory T cells and myeloid-derived suppressor cells)
are not the only factors being evaluated regarding their potential role as prognostic
and/or predictive factors. Many ongoing clinical trials are exploring the activity of
immune checkpoint modulators in BC treatment, both in the advanced and neo-
adjuvant setting. Although this field is expanding with exciting new discoveries and
promising clinical results—and creating great expectations—there remain many uncer-
tainties yet to be addressed satisfactorily before this long awaited therapeutic promise
can come to fruition.
ABBREVIATIONS
ADCC antibody-dependent cellular cytotoxicity
AEs adverse events
APC antigen-presenting cells
ARG1 arginase-1
BC breast cancer
CbPt carboplatin
CI confidence interval
CR complete response
CRT calreticulin
CTLs cytotoxic T cells
DAMPs damage-associated molecular patterns
dd dose dense
DLT dose-limiting toxicity
DOR duration of response
ER estrogen receptor
H&E hematoxylin and eosine
HER2 human epidermal growth factor receptor 2
HLA human leukocyte antigen
HMGB1 high-mobility group box 1
HR hormone receptor
IDO indoleamine-pyrrole 2,3-dioxygenase
IFN interferon
IHC immunohistochemistry
IL-10 interleukin-10
IL-2 interleukin-2
irAEs immune-related adverse events
LCK lymphocyte-specific kinase
LPBC lymphocyte predominant breast cancer
MAb monoclonal antibody
Breast Cancer Immunology and Immunotherapy 3
MBC metastatic breast cancer

MDSCs myeloid-derived suppressor cells
MHC major histocompatibility complex
mTNBC metastatic triple negative breast cancer
NK natural killer
NO nitric oxide
NSCLC non-small cell lung cancer
ORR overall response rate
OS overall survival
pCR pathological complete response
PD progression of disease
PD-1 programmed cell death protein 1
PFS progression-free survival
PR partial response
ROS reactive oxygen species
RP2D recommended phase 2 dose
RT radiotherapy
SD stabilization of disease
TCR T-cell receptor
TGF-β transforming growth factor β
TH1 T helper type 1
TH2 T helper type 2
TILs tumor-infiltrating lymphocytes
TN triple negative
TNBC triple negative breast cancer
Treg regulatory T cells
VEGF vascular endothelial growth factor
1. INTRODUCTION
It is now known that solid tumors, such as breast tumors, originate
from single cells and evolve to malignant masses of invasive tissue taking
advantage of the body’s innate inflammatory response, while evading the
adaptive immune system (Quigley and Kristensen, 2015). Thus, the immune
system plays an integral and complex role in breast cancer (BC) biology,
both promoting tumor growth and mediating the eradication of disease.
This dual role is based on the dynamic interplay between various immune
effector cells, tumor cells, stromal cells, and soluble factors (Cimino-
Mathews et al., 2015). This complex interplay between the immune system
and cancerous cells is now considered a new hallmark of cancer (Hanahan
and Weinberg, 2011). The tumor and the immune system interact through
complex events resulting in either tumor detection, followed by tumor
eradication, or immune system evasion by the tumor (Becker et al., 2013).

According to this hypothesis, tumor variants unable to evade the immune
system’s cancer surveillance are eradicated, whereas variants with the genetic
advantage of remaining undetected are selected through natural selection.
The notion that tumors develop mechanisms to avoid detection by the
immune system was also based on the observation that tumors with low
human leukocyte antigen (HLA) class I expression showed reduced infiltra-
tion with CD8+ T cells (Whitford et al., 1992). The ability of CD8+ and
CD4+ T cells to recognize unique tumor antigens has also been reported
(Baxevanis et al., 1994; Dadmarz et al., 1995). However, selective immune
pressure on tumor cells can result in the buildup of those with those abnor-
malities which predispose them to escape immune surveillance. These
anomalies may include loss of major histocompatibility complex (MHC)
class I protein expression, as well as other defects in antigen presentation pro-
cesses, T-cell receptor (TCR) signaling, and interferon (IFN) signaling path-
ways and loss or mutation of tumor antigens (DeNardo and Coussens, 2007;
Singh and Paterson, 2007).
T cells are the primary means by which the immune system attacks
tumors. Generally, CD8+ cytotoxic T cells (CTLs) and CD4+ helper
T (Th) 1 cells prevent cancer development via mechanisms commonly
involving production of interferon IFN-γ and cytotoxins (Zamarron and
Chen, 2011). These activated T cells expand and home to the tumor micro-
environment, where they facilitate tumor cell death. The protective role of
the adaptive immune system is now termed immune surveillance, and
tumors’ attempt at hiding from the immune system by concealing their anti-
gens or shutting down immune cell effectors functions is called immune
editing (reviewed in Schreiber et al., 2011).
Finally, tumors attain complete autonomy from immune surveillance
achieving the escape phase. This tumor phase is achieved by making
tumor cells, as well as immune cells within the tumor microenvironment,
upregulate inhibitory immune checkpoint pathways, such as the pro-
grammed cell death protein 1 (PD-1) pathway. Other key mechanisms
actively promoting tumor growth include the inhibition of tumor antigen-
specific T cells by intratumoral myeloid-derived suppressor cells (MDSCs),
T regulatory cells (Tregs), a change from a T-helper type 1 (TH1) to a
T-helper type 2 (TH2) protumorigenic immune response and production
of soluble factors that, apart from inhibiting T-cell and dendritic function,
promote stromal remodeling and angiogenesis (Dunn et al., 2002; Swann
and Smyth, 2007).
2. MECHANISMS OF THE IMMUNE RESPONSE

INHIBITION IN BC: ROLE OF MDSCs AND
REGULATORY T CELLS
BC cells are known to develop a variety of strategies to escape recog-
nition by immune cells and to suppress immune reactions. In addition, tumor
cells may induce the conversion of immune cells into supporters of tumor
growth during disease progression (Ohara et al., 2009). These important
effects in the tumor microenvironment are mediated by two different mech-
anisms: cell–cell contacts and secretion of factors such as cytokines. Recently,
the immune microenvironment in BC has been found to be very heteroge-
neous and to be affected by hypoxia-related genes (Duechler et al., 2014).
Based on animal models, previous research concludes that close to all inva-
sive cancers are successful at activating abnormal myelopoiesis and recruiting
immature CD11b+GR-1 myeloid cells with immune suppressive function
into different tissues (Gabrilovich et al., 2012). These cells are known as
MDSCs. Even though human MDSCs are less defined, they are generally
considered to be myeloid-derived, nonlineage determined and with poor
Ag presentation capacity. Thus, MDSCs are often described with negative
markers such as HLA-DR negative, and positive markers such as CD33+
and CD11b+, with alternative markers for different phenotypes, such as
CD14+ (monocytic MDSCs) or CD15+ (granulocytic MDSCs). This cate-
gorization accounts for the heterogeneity of MDSCs reported in cancer
research (Solito et al., 2014). In this line, MDSCs comprise a heterogeneous
population of myeloid cells, including myeloid progenitor cells, immature
dendritic cells, and granulocytes. MDSCs are found not only in cancer but also
in inflammatory and infectious processes and are characterized by an extraor-
dinary capacity to suppress T-cell responses (Gabrilovich and Nagaraj, 2009).
In cancer development, the inhibitory effects of MDSCs on innate and
adaptive immunity result in the hindrance of immune surveillance and pre-
vention of the immune system from eliminating newly transformed cells
(Stewart and Abrams, 2008). BC patients present elevated levels of MDSCs
and the highest are found in patients with metastatic disease (Diaz-Montero
et al., 2009; see Markowitz et al., 2013 for a review of MDSCs’ role in BC).
To make matters worse, a distinct characteristic of MDSCs is that they can
induce certain responses to cancer treatments by manipulating negative reg-
ulatory factors of the patients’ immune pathways to generate chemoresistant
niches (Jinushi et al., 2013).
MDSC induction is likely caused by a general inflammatory process in

BC (Jiang and Shapiro 2014) and can be associated to a wide variety of cyto-
kines (Gabrilovich et al., 2012). Factors that induce MDSC expansion
include GM-CSF, PGE2, IL-6, stem cell factor, and vascular endothelial
growth factor (VEGF), while IFN-γ ligands of toll like receptors, IL-13,
IL-4, and transforming growth factor β (TGF-β) are associated with MDSC
activation (Gabrilovich and Nagaraj, 2009). Among these factors, GM-CSF
is considered one of the primary BC-derived soluble factors involved in the
differentiation of monocytic/granulocytic progenitor cells into MDSCs
(Gabrilovich et al., 2012).
Mechanisms of MDSC inhibitory action include arginase-1 (ARG1)-
mediated local depletion of arginine and cysteine. The depletion of these
amino acids causes downregulation of the ζ-chain in the TCR complex
and proliferative arrest of antigen-activated T cells (Rodriguez et al.,
2004; Srivastava et al., 2010). MDSC action is also mediated by the inducible
nitric oxide synthase (iNOS), which oxidizes arginine generating (nitric
oxide) NO and citrulline, and by NADPH oxidase-dependent production
of reactive oxygen species (ROS) and reactive nitrogen species (Corzo et al.,
2009). The molecular effects of reactive species in T cells are several and may
range from loss of TCR ζ-chain expression (Schmielau and Finn, 2001) to
desensitization upon nitration of the TCR (Nagaraj et al., 2007). MDSCs
also induce nitration of MHC class I molecules on BC cells, making them
unable to effectively present specific peptide, and thus rendering tumor cells
resistant to antigen-specific CTLs. MDSCs also induce nitration of MHC
class I molecules on BC cells, leaving them unable to effectively present a
specific peptide, and thus rendering tumor cells resistant to antigen-specific
CTLs (Lu et al., 2011). Additionally, previous studies have shown the role of
indoleamine-pyrrole 2,3-dioxygenase (IDO), a rate-limiting enzyme in the
catabolism of tryptophan, on tumor-induced immunosuppression (Friberg
et al., 2002; Mellor and Munn, 2004; Uyttenhove et al., 2003). Thus,
upregulation of IDO-1 inhibits T cell activation and proliferation by tryp-
tophan deprivation (Hill et al., 2007). In this line, MDSC has recently been
found to suppress antitumor immune responses through IDO expression, a
process which correlates with lymph node metastasis in BC patients
(Yu et al., 2013).
MDSCs also produce immunosuppressive cytokines, such as TGF-β and
IL-10. TGF-β stimulates bone metastases in BC patients and activates
epithelial-to-mesenchymal transition thus increasing tumor invasiveness
(Buijs et al., 2011). Moreover, MDSCs modulate alternatively activated M1

and M2 macrophages which not only create an inflammatory microenviron-
ment but can also act as mediators of tumor initiation, angiogenesis, and
metastasis (Murdoch et al., 2008; Shozaei et al., 2008).
Finally, there is another mechanism—via Treg cells—for MDSC’s
inhibitory effect on the immune system. Treg cells, whose numerical and
functional importance have yet to be clarified, are generated in the thymus
and the periphery. In the thymus, Treg cells originate roughly in sync with
the positive selection of conventional CD4+ T cells, whereas in the periph-
ery, a number of triggers induce the expression of FoxP3 in T cells. MDSCs
stimulate the clonal expansion of antigen-specific TReg cells, promote
CD4+ T-cells conversion into induced TReg cells. This conversion involves
cell-to-cell contact (including CD40–CD40L interactions) (Pan et al., 2010),
the production of soluble factors (such as IFN-γ, IL-10, and TGFβ)
by MDSCs (Huang et al., 2006), and probably also the expression of ARG1
by MDSC (Serafini et al., 2008). Moreover, human MDSCs have been
found to promote the transdifferentiation of TH17 cells into FOXP3+
-induced Treg cells by producing TGFβ and retinoic acid (Hoechst et al.,
2011). This mechanism is potentiated by the effect of MDSC on macrophages
to M2 type, which also release TGF-β1 and IL-10, not only inducing Th2
differentiation and recruitment, but also mediating Treg development. This
process results in tumor development promotion through the inhibition of
anticancer immune responses (Biswas and Mantovani, 2010; de la Cruz-
Merino et al., 2009). Thus, in human breast carcinomas, tumor-associated
macrophage (TAM) density correlates with poor prognosis. In fact, Laoui
and colleagues have shown that eliminating macrophages from mice BC
tumors, either via genetic or therapeutic means, results in retarded tumor pro-
gression (Laoui et al., 2011). In addition, Tregs can also be recruited from cir-
culation to BC sites through distinct chemotactic mechanisms including
CCL22/CCR4, CXCL12 (SDF-1)/CXCR4, and CCL5 (RANTES)/
CCR1 axes (Gobert et al., 2009; Tan et al., 2011; Yan et al., 2011). Finally,
tumor-evoked regulatory B cells can directly convert resting CD4+ T cells
into FOXP3-expressing Treg cells in a TGF-β-dependent manner
(Olkhanud et al., 2011).
The role of Tregs in the immunopathogenesis of BC has previously
been reviewed (Watanabe et al., 2010). As stated earlier, the precise mech-
anisms by which Tregs suppress immune cell functions remain unclear,
but there is evidence of both direct inhibition through cell–cell contact
and indirect inhibition through the secretion of antiinflammatory media-

tors (Watanabe et al., 2010). High Treg cell levels have been reported in
peripheral blood, lymph nodes, and tumor specimens from patients with
different types of cancer. Infiltration of FOXP3-positive Tregs into BC
tumors, which is associated with more aggressive tumors, has been observed
in numerous studies. Consequently, it has been strongly suggested that the
increase in functional Treg cell levels in cancer patients might be seen as a
response to malignant transformation (Beyer et al., 2006). Thus, quantifying
Tregs has become a valuable tool in the assessment of BC progression
and prognosis (Bohling and Allison, 2008; Liyanage et al., 2002;
Ohara et al., 2009).
Tregs found in cancer patients’ tumors and peripheral circulation have
distinctive properties. Local immune responses are controlled by Tregs that
are induced in the tumor microenvironment. Tregs present in cancer
patients are, by and large, adaptive or inducible Tregs (iTregs) which differ
from thymus-derived natural Tregs (nTregs) which are responsible for
the maintenance of peripheral tolerance in healthy donors (Whiteside
2012). Moreover, several subsets of Treg cells have been identified and
characterized, such as CD8+ Treg cells, CD4+ Treg cells, and γδ-TCR
(Wang, 2008). The conversion mechanism CD25 CD4+ in CD25+
CD4+ in the periphery, in vitro, involves TGF-β in a murine model
(Feuerer et al., 2009). Finally, IDO may also induce FoxP3 expression in
CD4 T cells and convert human and murine CD4+CD25 T cells to
CD4+CD25+FoxP3+ cells (Curti et al., 2007).
Even though some authors consider FoxP3 protein the most important
Treg marker (Fontenot et al., 2003), FoxP3 does not necessarily confer a
Treg phenotype when expressed in CD4+ T lymphocytes (Gavin et al.,
2006). Moreover, only approximately half the CD4+CD25+ population
expresses FoxP3 (Roncador et al., 2005). Thus, CD127 (IL-7 receptor)
was later identified as a suitable negative marker for Tregs. CD127 is absent
in Tregs and its expression inversely correlates with Foxp3 expression,
together with other markers, such as CD25. The T cells CD127low/
CD25high identify over 95% of the Foxp3+ cells in peripheral blood
(Liu et al., 2006; Seddiki et al., 2006).
Although the exact mechanisms of Treg suppression of the immune
response remain unknown, the effect seems to be largely dependent on
the expression of the transcription factor FOXP3 which controls some genes
encoding proteins like CD25, GITR, and CTLA-4 among others, capable
of mediating Treg suppressive functions (Gavin et al., 2007; Zheng et al.,

2007). In addition, FOXP3 inhibits production of effector cytokines like
interleukin-2 (IL-2) after TCR stimulation of T cells (Vignali et al.,
2008). Other mechanisms of immunosuppression involve direct cell-to-cell
contact with antigen-presenting cells (APC) via TGF-β or CTLA-4 and
secretion of immunosuppressive cytokines such as interleukin-10 (IL-10),
and TGF-β, among others (Collison et al., 2009; de la Cruz-Merino
et al., 2009). Therefore, FOXP3-expressing Treg cells are potent mediators
of peripheral immune tolerance, suppressing a wide range of immune cells,
including CD4+ and CD8+ T cells, natural killer (NK) cells, NKT cells,
B cells, and APC, through inhibiting target cell activation and proliferation
as well as effector functions (Sakaguchi et al., 2010; Shevach, 2009).
3. IMMUNE SYSTEM IN THE THERAPEUTIC RESPONSE

IN BC. THE IMMUNOGENIC CELL DEATH
Activation of the immune system could mediate the antitumor effects of
the conventional anticancer agents, including cytotoxic drugs, radiation ther-
apy, antibody-based therapy, targeted therapies (Andre et al., 2013; Apetoh
et al., 2007; Denkert et al., 2011, Galluzzi et al., 2013; Vatner et al., 2014),
as well as immune checkpoint blockers (Lesokhin et al., 2015). These
treatments could provoke immunogenic cancer cell death, which would sub-
sequently activate the immune system (Galluzzi et al., 2015). Thus, immuno-
genic cell death constitutes a prominent pathway for the activation of the
immune system against cancer, which in turn determines the long-term
success of anticancer therapies (Kroemer et al., 2013).
Cells may experience various forms of death, each resulting in different
antigenic presentation and clearance mechanisms (Galluzzi et al., 2007).
Physiological cell death, which occurs as a continuous byproduct of
cellular turnover, frequently occurs through apoptosis, a cell death widely
considered as nonimmunogenic or even tolerogenic, thereby avoiding auto-
immunity (Tesniere et al., 2008). This homeostatic cell death, which often
occurs through apoptosis, can be considered as tolerogenic if it promotes
tolerance to the self or as null, in the case of not presenting any impact
on the immune system (Galluzzi et al., 2012; Green et al., 2009). Neverthe-
less, the concept that considers apoptosis as tolerogenic and necrosis as
immunogenic is not completely exact (Tesniere et al. 2008; Zitvogel
et al., 2004). Thus, it seems that apoptosis is a nonuniform process in
biochemical terms, i.e., distinct molecular pathways can lead to cell death
and induce stimulus-specific alterations (Kroemer and Martin, 2005).
The question is, then, how does the immune system distinguish between
immunogenic and nonimmunogenic cell death modalities. Dying, stressed,
or injured cells release or expose molecules on their surface that can function
as either adjuvant or danger signals for the innate immune system
(Matzinger, 1994). These signals were later called damage-associated molec-
ular patterns (DAMPs) (Krysko et al., 2012).
Immunogenic cell death involves changes in the composition of the cell
surface as well as the release of soluble mediators, all following a specific tem-
poral sequence (Kroemer et al., 2013). This immunogenic death is character-
ized by a preapoptotic surface presentation or release of cryptic antigens, the
DAMPs, which are a compilation of molecules sequestered under homeo-
static conditions within various subcellular compartments (Garg et al.,
2013). These factors include preapoptotic exposure of calreticulin (CRT)
and other endoplasmic reticulum proteins at the cell surface, the secretion
of ATP during the blebbing phase of apoptosis, and the cell death-associated
release of the nonhistone chromatin protein high-mobility group box 1
(HMGB1) (Apetoh et al., 2007; Ghiringhelli et al., 2009; Obeid et al., 2007).
In response to multiple immunogenic cell death inducers, such as anthra-
cyclines and ionizing irradiation, a fraction of CRT translocates from the endo-
plasmic reticulum lumen to the surface of stressed and dying cancer cells (Gardai
et al., 2005; Obeid et al., 2007). This phenomenon occurs before cells expose
phosphatidylserine on the outer leaflet of the plasma membrane. Conversely,
anthracycline-induced CRT exposure does require the formation of ROS
and NO (De Boo et al., 2009). When elicited by anthracyclines or oxaliplatin,
the exposure of CRT is activated by an endoplasmic reticulum stress response
that involves the phosphorylation of the eukaryotic translation initiation factor
eIF2α by the PKR-like endoplasmic reticulum kinase (PERK).
CRT exposure constitutes one of the major checkpoints determining
the immunogenicity of cell death, allowing the phagocytosis of the cell
by macrophages and dendritic cells via interaction with CD91 (LRP1)
(Kroemer et al., 2013).
The second signal of immunogenic cell death is the release of ATP, a
mechanism dependent on autophagy (Michaud et al., 2011), resulting in
a chemoattractant signal for machrophages and dendritic cells interacting
with P2-Y2 and P2RX7 (via activation of the NLRP3 inflammasome)
receptors (Muller et al., 2010; Zitvogel et al., 2012).
The third signal of immunogenic cell death is the release of HMGB1, an

abundant nuclear nonhistone chromatin-binding protein (Garg et al., 2010).
HMGB1 is actively secreted by cells of the innate immune system in
response to pathogenic products (in the absence of cell death) and released
by injured cells as they succumb to primary or secondary (postapoptotic)
necrosis (Andersson and Tracey, 2011). HMGB1 triggers proinflammatory
responses by signaling through receptors on the surface of both immune and
nonimmune cells. More specifically, HMGB1 can bind to TLR4, thus
activating the release of proinflammatory cytokines by monocytes/
macrophages, as well as to RAGE, hence modulating endothelial and cancer
cell functions (Sims et al., 2010). When administered to TLR4-expressing
dendritic cells in vitro, HMGB1 augments the expression of pro-IL-1β
(Apetoh et al., 2007).
Finally, monoclonal antibodies (MAbs), used effectively as molecule-
specific targeting drugs for cancer treatment, may also facilitate the liberation
of tumor-associated antigens through Fc-mediated antibody-dependent cellu-
lar cytotoxicity (ADCC), complement-dependent cytotoxicity, complement-
dependent cellular cytotoxicity, opsonization, and phagocytosis mechanisms
and enhance antigen presentation function (Haynes et al., 2008).
Therefore, we now know that different cancer treatments possess the
capacity to trigger tumor antigen release and danger signals, which engage
both the innate and adaptive immunity. Thus, the immunologic death of
cancer cells as well as the immune response of the patients upon cancer treat-
ment needs to be further characterized and monitored in order to gain insight
into the molecular mechanisms and pathways of the therapeutic response.
This knowledge would also lead to new markers of cancer evolution and puta-
tive predictors of response to therapy in the clinical management of BC.
4. T-INFILTRATING LYMPHOCYTES MOLECULAR AND

GENOMIC CHARACTERIZATION AS A STRATEGIC
TOOL TO BETTER UNDERSTAND BC BEHAVIOR
Immunosurveillance, when applied to cancer, describes the fine mod-
ulation of the host immune response toward neoplastic cells (Corthay,
2014). This interplay between immune and transformed cells (i.e.,
immunoediting) shapes the features of the tumoral mass which may finally
progress with no control in the event of ultimate failure of immunological
barriers (Mittal et al., 2014).
In BC, as in many other tumor types, immunosurveillance is functionally

represented by the presence in the neoplastic cellular mass of “tumor-
infiltrating lymphocytes” (TILs). TILs in breast tumors were observed for
the first time in 1922 and even then their presence was associated to positive
outcomes in a clinical cohort (Sistrunk and Maccarty, 1922). This was later
confirmed by Aaltomaa et al. (1992) who described the existence of a
lymphocyte-rich phenotype of breast tumor followed-up for over 10 years.
This positive correlation has been confirmed in many more studies since
then, but only recently a deeper functional characterization of TILs has
started to be considered as a putative prognostic and predictive biomarker
(Savas et al., 2016) (Tables 1 and 2). International efforts to define a consen-
sus for TILs evaluation in BC led to The International TILs Working Group
to recently describe the term lymphocyte predominant breast cancer
(LPBC) phenotype for tumors with very high immune infiltrate, i.e., over
50–60% of stromal surface area occupied by TILs (Salgado et al, 2015). This
LPBC is present in 20–28% of patients (Loi et al., 2013). TILs can be stromal
or intratumoral lymphocytes. Stromal lymphocytes are dispersed within the
stroma surrounding carcinoma cells but not in direct contact with them,
whereas intratumoral lymphocytes are in direct contact with tumor cells
(Salgado et al., 2015). Due to the dynamic and changing nature of tumor
microenvironment, this “static” distinction may look somehow artificial
and more related to what can be observed in histological slides. Neverthe-
less, it is commonly accepted that stromal TILs are a better and more repro-
ducible parameter than intratumoral ones. The International TILs Working
Group has developed a standardized methodology to improve consistency
and reproducibility in the measurement of TILs by visual assessment on
hematoxylin and eosine (H&E) sections for daily clinical and research prac-
tice (Salgado et al., 2015). The working group concluded that TILs should
be reported for the stromal compartment (% stromal TILs), considering a full
assessment of average TILs in the area within the borders of the invasive
tumor, at least in one section of 4–5 μm. The working group suggested that
the quantitative assessment of TILs by digital image analysis (TILs per mm2
stromal tissue) might be useful for standardization in the future. Full sections
are preferred over biopsies, although cores can be used in the prethera-
peutic neoadjuvant setting. There is currently no validated methodology
developed to score TILs after neoadjuvant treatment. The working group
reckoned that TILs are bound to provide more relevant biological informa-
tion when measured as a continuous variable; however, it admits there is no
formal recommendation for a clinically relevant TIL threshold(s) yet.
Table 1 Main Clinical Studies Analyzing pCR-Predictive Role of TILs Upon Neoadjuvant Chemotherapy in Breast Cancer
Study Design Therapy N Measure Result References
Retrospective Anthracycline– 73 p53, PgR, Ki67, CD3, CD83, CD3 and CD83 correlate with pCR Hornychova
taxane-based CD68+ (IHC) et al. (2008)
Retrospective, Anthracycline– 1058 TILs markers (IHC and Intratumoral TILs and LPBC correlate Denkert et al.
GeparDuo/Trio taxane-based RT-PCR) with pCR. Inflammatory genes expression (2010)
vinorelbine/ linked to the presence of TILs
capecitabine
(GeparTrio)
Retrospective, ER-, Anthracycline- 113 High/low TILs tumors definition, TILs correlate with response to West et al.
EORTC10994/ or taxane-based TILs gene expression signature anthracycline-based therapy (2011)
BIG01 and markers (IHC in TMA)
Retrospective Anthracycline-, 474 TILs, basal, and apoptosis TILs and apoptosis markers correlate with Ono et al.
cyclophosphamide-, markers (IHC) pCR in TN subtype (2012)
or taxane-based
Retrospective Anthracycline– 68 TILs markers (IHC), TNM TILs best predictor for pCR; Yamaguchi
taxane-based staging, histological grade T-lymphocites are better predictors than et al. (2012)
B-lymphocites
Retrospective, Anthracycline with or 996 Public database for STAT1 gene High immune module scores Ignatiadis
8 cohorts without taxane-based module and immune response were associated with increased pCR et al. (2012)
gene expression probability in all BC subtypes
Retrospective Anthracycline– 180 CD8, FOXP3, IL17F (IHC) CD8+ and FOXP3+ lymphocites Oda et al.
taxane-based correlate with pCR (2012)
Retrospective Anthracycline– 1810 B-cell derived metagene Ig κ C (IGKC) as a single marker Schmidt et al.
taxane-based (RNA and protein levels) is similarly prognostic and predictive (2012)
to neoadjuvant chemotherapy as the entire
B-cell metagene
Continued
Table 1 Main Clinical Studies Analyzing pCR-Predictive Role of TILs Upon Neoadjuvant Chemotherapy in Breast Cancer—cont’d
Retrospective Anthracycline– 153 CD8, CD4, FOXP3 (IHC); CD8, CD4, FOXP3 correlated with pCR; Seo et al.
taxane-based BCSC phenotype, EMT markers CD8+-independent predictive factor for (2013)
pCR
Prospective, Anthracycline– 313 Intratumoral/stromal TILs (H&E) Stromal TILs and LPBC correlated with Issa-
HER2-, taxane-based pCR Nummer
GeparQuinto et al. (2013)
Retrospective, TN Anthracycline– 304 Intratumoral/stromal TILs (H&E) TILs in residual disease after neoadjuvant Dieci et al.
taxane-based chemotherapy are associated with better (2014)
prognosis
Retrospective, TN Anthracycline– 175 TILs, CD8, CD4, FOXP3 TILs, CD8, CD4, FOXP3 correlate with Lee et al.
taxane-based pCR (2013)
Retrospective, TVA Anthracycline– 47 CD8, FOXP3 (IHC) CD8, FOXP3 correlated with pCR Nabholtz
taxane- et al. (2014)
based + panitumumab
Retrospective, Carboplatin and 580 TILs/LPBC (H&E); immune- TILs predict pCR; immunologic factors Denkert et al.
HER2+, and TN, anthracycline– activating and suppressive factors were highly significant predictors of pCR (2015)
GeparSixto taxane-based (mRNA analysis)
Retrospective, Trastuzumab, 455 TILs (H&E) Presence of TILs at diagnosis is an Salgado et al.
HER2+, lapatinib, independent, positive, prognostic marker (2015)
NeoALTTO or the combination for both pCR and survival end-points
followed by paclitaxel
Retrospective Anthracycline–taxane 177 IHC assessment of CD8+/FOXP3+ CFR correlates with pCR Asano et al.
-based TIL ratio (called “CFR”) (2016)
BC, breast cancer; BCSC, breast cancer stem cells; EMT, epithelial–mesenchymal transition; ER, estrogen receptor; IHC, immunohistochemistry; HER2, human epidermal growth factor
receptor 2; H&E, hematoxylin–eosin; LPBC, lymphocytes predominant breast cancer; N, Study’s sample size; pCR, pathological complete response; RT-PCR, reverse transcription
polymerase chain reaction; TILs, tumor-infiltrating lymphocytes; TMA, tissue micro array; TN, triple negative; TNM, tumor-node-metastasis classification of malignant tumors.
Table 2 Main Clinical Studies Analyzing Outcome-Predictive Role of Tumor-Infiltrating Lymphocytes (TILs) Upon Adjuvant Chemotherapy in Breast Cancer
Retrospective, Anthracycline-based 255 CD3, CD8, CD4, CD20, AC-based therapy associated with West et al.
EORTC10994/BIG01 TIA-1 (IHC) increased DFS only in patients with high (2011)
levels of intraepithelial CD3+ TILs
Retrospective Anthracycline–taxane- 1334 Intratumoral/stromal/ In multivariate analysis, total CD8+ T-cell Mahmoud
based distant CD8+ TILs (IHC) count was an independent prognostic et al. (2011)
factor, HR 0.58 (BCSS)
Retrospective, high-risk pts Standard systemic therapy 3403 Intratumoral/stromal CD8+ TILs are an independent Liu et al.
CD8+ TILs (IHC) prognostic factor associated with better (2012)
survival in basal-like TNBC, but not in
nonbasal TNBCs nor in other intrinsic
molecular subtypes
Retrospective, BIG 02-98 Anthracycline- or 2009 Intratumoral/stromal TILs In TN (N ¼ 256), increasing TILs in the Loi et al.
trial anthracycline–taxane- (H&E) stroma was associated with excellent (2013)
based prognosis HR 0.84 (DFS); 0.82 (OS)
Retrospective, TN, ECOG Anthracycline- or 481 Intratumoral/stromal TILs Stromal TILs constitute a robust Adams et al.
E2197, and E1199 anthracycline– (H&E) prognostic factor HR 0.86 (DFS); 0.81 (2014)
taxane-based (OS)
Prospective/retrospective, Anthracycline-based 935 Intratumoral/stromal TILs In TNBC (N ¼ 134), stromal TILs Loi et al.
FinHER therapy + docetaxel or (H&E) significantly associated with lower risk of (2014)
vinorelbine (+trastuzumab distant recurrence, HR 0.79 (DFS) and
in HER2 +) 0.80 (OS). In HER2+ BC (N ¼ 209),
direct association between TILs and lower
risk of recurrence, HR 0.77 (DFS)
Continued
Table 2 Main Clinical Studies Analyzing Outcome-Predictive Role of Tumor-Infiltrating Lymphocytes (TILs) Upon Adjuvant Chemotherapy in Breast Cancer—
cont’d
Retrospective Variable 12439 CD8, FOXP3 (IHC) CD8 in stroma and tumor correlate with Ali et al.
28–21% reduced risk of BCSS, greater in (2014)
ER BC
Retrospective Not specified 636 PDL1 (mRNA PDL1 associated with better RFS and Schalper
fluorescence TILs et al. (2014)
hybridization), TILs
(H&E)
Retrospective Anthracycline-based 781 Intratumoral/stromal TILs TILs have prognostic role in TNBC and Dieci et al.
(H&E) suggested prognostic impact on HER2+ (2015)
tumors
Retrospective, HER2 + Anthracycline–taxane- 447 TILs (H&E), TLS, MHC TILs are independent prognostic factor in Lee et al.
based + trastuzumab I (IHC) HR/HER2+ BC (2015)
Prospective/retrospective, Anthracycline–taxane- 2618 Stromal TILs (H&E) TILs are prognostic factor in BC Kotoula
HeCOG 10/97-0005-08 based et al. (2016)
AC, adriamycin/cyclophosphamide; BCSS, breast cancer-specific survival; DFS, disease-free survival; ER, estrogen receptor negative; HER2, human epidermal growth factor receptor 2;
H&E, hematoxylin–eosin; HR, hazard ratio; HR, hormone receptor negative; IHC, immunohistochemistry; MHC, major histocompatibility complex; N, Study’s sample size; OS, overall
survival; pts, patients; RFS, not defined; TN, triple negative; TNBC, triple negative breast cancer; TLS, tertiary lymphoid structures.
Finally, the working group’s guidelines underscore the importance of not

using TILs levels to withhold adjuvant chemotherapy or trastuzumab
therapy in triple negative (TN) and HER2+ BC, respectively.
Evidence suggests that TILs can be easily observed in histological sections
upon H&E staining (Savas et al., 2016), and there seems to be associations
between their levels, disease prognosis, and response to neoadjuvant treat-
ments in TN (ER/PgR/HER2) and HER2+ BC subtypes (Ahn et al.,
2015). The statistical correlation between lymphocytic infiltration and path-
ological response upon neoadjuvant chemotherapy in TN breast cancer
(TNBC) has been described by Ono et al. (2012). The authors reported that
TILs score was significantly higher in TN and HER2+/HR subtypes than
in HER2/HR+ subtype. Further, the pathological complete response
(pCR) rate was significantly higher in TNBC with high TILs scores than
in TNBC with low scores (37% vs 16%, p ¼ 0.05). Similar results were
observed in the BIG 02-98 adjuvant trial, comparing doxorubicin plus
docetaxel or doxorubicin in monotherapy, where patients with ER/
HER2 tumors and high percentage of TILs had a 5-year survival rate sig-
nificantly higher than those with a lower grade of infiltration (92% vs 71%,
hazard ratio 0.39 vs 1.87, respectively) (Loi et al., 2013). In patients with
HER2+ tumors, positive correlation between TILs and disease-free survival
was reported exclusively for the anthracycline-only treatment arm.
The association between pCR upon neoadjuvant treatment and presence
of TILs has been described in several studies (Table 1). Mao et al. (2014), in
their meta-analysis of 13 clinical trials and retrospective studies, showed
that the presence of TILs in tumor biopsies could increase four times the
pCR rate, independently from their location (stromal, intraepithelial, or
both), but only in TN and HER2+ cancers, not in estrogen receptor (ER)+
ones. Independent studies have confirmed that a low pCR rate is associated to
TILs in ER+ and HER2 breast tumors (Bhargava et al., 2010; Tan et al.,
2009). Among the studies included in the meta-analysis by Mao et al.
(2014), the neoadjuvant anthracycline/taxane-based clinical trials GeparDuo
(a randomized phase III clinical trial comparing docetaxel in dose dense
combination with doxorubicin vs a sequential schedule of doxorubicin/
cyclophosphamide followed by docetaxel) and GeparTrio (randomized
phase III clinical trial to evaluate pCR after four to six additional cycles
of docetaxel, doxorubicin, and cyclophosphamide (TAC) in patients respon-
ding to an initial two cycles of TAC or four cycles of TAC followed by four
cycles of vinorelbine and capecitabine for nonresponders to the initial two
cycles of TAC) showed a 40% rate of pCR in patients with high lymphocytic
infiltration, but only 7.2% in those with no infiltration (Denkert et al., 2010).
These results have been recently confirmed in the PREDICT study for
patients with HER2 BC as part of the GeparQuinto neoadjuvant trial (ran-
domized phase III clinical trial exploring the integration of bevacizumab,
everolimus, and lapatinib into standard neoadjuvant chemotherapy regi-
mens). Multivariate analysis results showed that stromal TILs were an inde-
pendent predictor of pCR levels, whereas intratumoral TILs independently
predicted pCR levels only at the univariate level (Issa-Nummer et al., 2013).
In recent years, TILs in BC have been phenotypically characterized in
relation with outcome and treatment efficacy. TILs observed in BC biopsies
are, for the most part, helper T cells (CD3+/CD4+), CTLs (CD3+/
CD8+), and Tregs (CD3+/CD25+/FOXP3+). CD8+ T cells, crucial ele-
ments of tumor-specific adaptative immune response, have been associated
with patient survival in various cancers, including colorectal and ovarian
cancers (Galon et al., 2006; Sato et al., 2005). CD4+ T cells include a broad
family of lymphocytes that can potentially promote tumor eradication, pro-
vide long-term immunity, and facilitate the reestablishment of immune cell
homeostasis, via the production of specific cytokines and through direct
cytotoxic effects on tumor cells (Dobrzanski, 2013). CD25+/FOXP3+
Treg cells’ function is to disrupt antitumor immunity, in order to avoid
unnecessary tissue damage, by suppressing the effect or functions of various
immune cells (e.g., cytokine production, activation, and proliferation of
CD4+ and CD8+ T cells). These Tregs are also implicated in
immunosurveillance escape of cancer cells (Ding and Zhou, 2012).
TILs composition in cancer has been shown to change upon treatment
and to vary its correlation with pCR (Seo et al., 2013). This observation
implies that the efficacy of any chemo- or radiotherapy is intrinsically deter-
mined by tumor-specific immune response. Turning things around, the
ability of tuning the innate and induced response of the immune system
offers a new and intriguing immunotherapeutic strategy. Thus, the identi-
fication of immune biomarkers and their characterization is a crucial step to
set up new, patient-tailored immunotherapies focused on the maintenance
and/or strengthening of patient’s immune response.
The presence of CD8+ T cells correlates with favorable prognoses
(Mahmoud et al., 2011). Upon interaction with their tumor targets, these
T cells produce IFN-γ which provokes tumor cell cytostasis and cell death
by cell cycle inhibition, apoptosis, angiostasis, and induction of macrophage
activity. Conversely, it has been described that a decrease of FOXP3+ Tregs
levels upon neoadjuvant therapy is associated with higher pCR (Liu et al.,
2012). The fact that low levels of FOXP3+ Tregs are associated with better
outcomes has been confirmed by the observation that high CD8+ T cells/
FOXP3+ Tregs TILs ratio predicts a favorable prognosis in primary tumors;
however, the association is reversed in metastatic disease (Cimino-Mathews
et al., 2013). This apparently discordant finding may be explained by taking
into account that the influence of CD8+ T cells and FoxP3+ Tregs may be
different in primary and metastatic breast tumors, since the FoxP3+ Tregs
have also been shown to exert a prosurvival influence in tumors with low
numbers of CD8+ T cells (Alexandrov et al., 2013). A classification system
including the CD8+/FOXP3+ ratio has even been proposed, since very
high rates of long-term relapse-free and overall survival (OS) have been
observed in patients scoring high in this ratio (Ladoire et al., 2011). In post-
treatment primary tissues high FOXP3+ Tregs levels predict lower pCR
levels, especially in the absence of CD8+ T lymphocytes (Liu et al.,
2012; Mao et al., 2014).
As aforementioned, as tumor evolves, the constant interplay between
immune response players and tumor microenvironment leads to a modifi-
cation of infiltrate composition. Other T cell subsets, such as CD3+/
CD4+/CD20+, have been characterized in tumor lymphocytic infiltration
associated with favorable prognoses (Brown et al., 2014; Gu-Trantien et al.,
2013; Hornychova et al., 2008). Moreover, the presence of immunosup-
pressive macrophages (CD68+/CD163+) is usually accompanied by poor
outcome (DeNardo et al., 2011; Richardsen et al., 2015; Tiainen et al.,
2015). These observations, when considered all together, emphasize once
again the importance of the immune context in determining clinical
outcome.
The success of neoadjuvant treatments mostly relies on the tumor’s own
immunological context and on the treatments’ impact on it. Neoadjuvant
therapies should be able to either stimulate an immune response in neoplastic
lesions where it is already activated or evoke the immune response where
it is absent or weak. In fact, several studies demonstrate that treatments
with taxanes or anthracyclines increase the number of TILs in TN tumors,
or stimulate lymphocytic infiltration where it was originally absent (Demaria
et al., 2011; Dieci et al., 2014). Such effects define TILs as a prognostic
factor of OS, especially when TILs are mainly composed of CD8 and
FOXP3-expressing lymphocytes T cells (Oda et al., 2012). Neoadjuvant
chemotherapy can alternatively modify the composition of lymphocytic
infiltrate, as shown by Hornychova and colleagues who reported an increase
of CD3+/CD56+ TILs and dendritic cells, together with a decrease of
monocytes and VEGF, in patients undergoing neoadjuvant treatment with

doxorubicin and paclitaxel (Hornychova et al; 2008).
With the purpose of improving cancer prognosis using gene expres-
sion profile data, several multigene tests have been developed as tools to
clinically determine the risk of relapse in early stage BC (Prat et al., 2012;
Sestak et al., 2013). More recent prognostic tests, such as EndoPredict
and Prosigna, are also able to predict early- and late metastases (Dowsett
et al., 2013, 2013; Parker et al 2009; Weigelt et al., 2012). Moreover,
Prosigna provides a classification of BC intrinsic subtypes (Luminal A,
Luminal B, HER2-enriched, and Basal-like) with proven clinical utility
in adjuvant therapy selection for early BC in medical practice. Some of
them, Prosigna (PAM50 assay), 21-gene recurrence score (Oncotype
DX), and Mammaprint (NKI-70) (Albain et al., 2010; Buyse et al., 2006;
Cuzick et al., 2011; Dowsett et al., 2010; Paik et al., 2004, 2006; van de
Vijver et al., 2002; van ’t Veer et al., 2002) have been shown to impact
treatment decisions (Albanell et al., 2012; Geffen et al., 2011; Lo et al.,
2010; Martı́n et al., 2015).
The potential of TIL levels as a putative prognostic biomarker for BC has
been explored also from the genomic point of view in order to analyze the
association between different expression profiles and disease outcome. Des-
medt and colleagues were among the first to provide evidence linking the
expression of immune response-related genes with a specific prognosis. In
an effort to classify tumor types according to their gene expression patterns
and correlate them with tumor outcome, they found that only the
expression of the immune response module correlated with survival in both
HER2+ and TNBC (Desmedt et al., 2008). The overexpression of a similar
immune-related gene module was associated with better prognosis, in
terms of reduced risk of distant metastasis, in a subset of ER tumors
(Teschendorff et al., 2008)
During the task of improving the definition of highly heterogeneous
TNBCs from a genomic point of view to better design targeted therapies,
Lehmann and collaborators identified an immunomodulatory tumor sub-
type. This subtype is characterized by the expression profile of specific
immune-related genes which improve relapse-free survival compared to
other genetic subtypes identified in their study (Lehmann et al., 2011).
Moreover, a possible relation between tumor proliferation and immune
gene signatures was investigated by Nagalla et al. (2013) who identified three
different immune metagenes (B/plasma cells, T/natural killer cells, mono-
cytes/dendritic cells). Their simultaneous expression was associated with
reduced risk of metastasis in Basal-like, Luminal B, and HER2-enriched

subtypes, especially in the highest proliferation tertiles. Interestingly,
reduced expression of only one of the three immune metagenes was related
to a worse prognosis, independently of the high expression of the other two.
This finding underlies the physiological importance of a complete response
(CR) to ensure a positive outcome.
Microarray analysis is another tool used to better define the associa-
tion between the expression of specific immune metagenes and prognosis
in different tumor subtype (Rody et al., 2009). This line of work
identified a lymphocyte-specific kinase (LCK) metagene marker that
showed a significant positive prognostic value in ER and ER+ HER2+
tumors. Expression of the same LCK marker together with another IgG
metagene also predicted a better response to neoadjuvant chemotherapy
in ER tumors.
Identifying relationships between immunogenic signatures and response
to therapy is another promising field to define the putative predictive value
of specific genetic patterns. For instance, analysis of genomic data regarding
response to anthracycline with or without taxane-based neoadjuvant che-
motherapy revealed that high expression of an immune response gene mod-
ule was associated with increased probability of pCR, in all cancer subtypes,
especially in HER2+ cancers (Ignatiadis et al., 2012). Recently, the expres-
sion of a specific subset of immune-related genes was shown to predict
improved response to trastuzumab treatment in HER2+ tumors, underlying
a functional relation between gene expression and the drug’s mechanism of
action (Perez et al., 2015).
In another study, gene expression profiles were assessed to measure
immune cell activation. These were correlated, together with a wider char-
acterization of immune cell infiltration, with response to trastuzumab-based
therapy in HER2+ BC (Salgado et al., 2015). More specifically, the authors
examined the interaction between changes in immune signatures, following
brief-exposure to trastuzumab, and achievement of pCR subsequent to pre-
operative trastuzumab and chemotherapy in HER2-positive. The investiga-
tors reported that significant increases in a signature of immune cell admixture
(Immune Index), observed only following brief-exposure to trastuzumab,
were predictive of response. These results confirm the potential clinical use
of immune genetic signatures as a predictive biomarker in HER2+ BC.
Trastuzumab mechanism of action has also been shown to work through
activation of ADCC by triggering FcγRIII on NK cells (Dubsky et al.,
2013). The correlation between NK-mediated ADCC and trastuzumab
clinical activity in HER2+ cancers has been explored by assessing clinical

and immunological response in patients receiving trastuzumab mono-
therapy as maintenance management after chemotherapy (Sestak et al.,
2013). At 6- and 12-month follow-ups, nonresponders—according to
Response Evaluation Criteria in Solid Tumors (RECIST) criteria—
presented significantly lower NK and ADCC activity than controls and
responders.
Data from the GeparSixto trial were analyzed in order to investigate
any relationship between immunological relevant genes and response to
carboplatin (CbPt) treatment in HER2+ and TNBC. When tumors
were divided into three groups by level of expression of the genes
under study, results showed that the group with the highest levels of
expression was also the one with best pCR rates (Denkert et al., 2015).
These results support the notion that CbPt induces an immunogenic type
of cell death.
5. NEOANTIGEN PREDICTION: GENOMIC TOOLS

FOR PERSONALIZED IMMUNOTHERAPY
Transformed cells, while showing a wide range of mutations, present
the intrinsic feature of expressing tumor-specific antigens (also known as
“neoantigens”). This characteristic opens the intriguing therapeutic oppor-
tunity of designing immunotherapies targeting these definite markers while
keeping, at least in theory, side effects at reduced levels (Lu and Robbins,
2016). The first and essential step toward taking advantage of the ability
to target tumor antigenic specificity relies on accurately defining the tumor.
The original and classical approach used peptides generated on the base of
the most common mutations of key genes (such as p53, k-RAS, BRAF)
and the stimulation of T cells in vitro (Bergmann-Leitner et al., 1998;
Ichiki et al., 2004; Sharkey et al., 2004). Nevertheless, the peculiarity of each
tumor implies that the majority of the mutations producing neoantigens are
tumor specific.
Recent advances in high-throughput sequencing techniques have
allowed a faster and more precise genomic characterization of the tumor.
Next-generation sequencing (NGS) allows the identification of alterations
in the whole genome as well as exome and transcriptome of a tumor.
A global view of the complexity and heterogeneity of tumor mutational
burden was provided by Alexandrov et al. (2013). In their work, they ana-
lyzed almost 5 million mutations from over 7000 tumors from which they
extracted 20 distinct mutational signatures, some of them present in different

tumors. The potential application in immunotherapy is evident as all
neoantigens derived from tumor-specific mutations may be targeted by
T cells, whose action can be boosted upon immune checkpoint blockade.
So far, existing evidence supports this strategy, especially in melanoma
and non-small cell lung cancer (NSCLC) models which show a high
mutational degree, according to Alexandrov and colleagues’ data. For
instance, in two independent cohorts of NSCLC treated with
pembrolizumab (an antibody targeting programmed cell death-1, PD-1),
higher nonsynonymous mutation burden in tumors was associated with
improved objective response, durable clinical benefit, and progression-free
survival (PFS) (Rizvi et al., 2015). A direct correlation between response to
anti-PD-1/PD-L1 therapies and wide mutational burden has also been
proposed by Champiat et al. (2014) based on data from different types of
solid tumors.
Similarly, patients with melanoma cancers with high mutational degree
experienced more favorable response to ipilimumab (anti-CTLA-4 anti-
body) treatment than similar patients with tumors with low mutational
degree (Snyder et al., 2014). van Rooij and colleagues reported the case
of a patient with stage IV melanoma who experienced clinical response
to ipilimumab treatment (van Rooij et al., 2013) and showed that the clinical
outcome was related to the increased T-cell reactivity against a mutant
epitope of ATR (ataxia telangiectasia and Rad3 related) gene product.
Tumor missense mutation profiles can also be subjected to in silico
analysis in order to predict potential high-affinity epitopes (Matsushita
et al., 2012). Fortunately, the patient’s HLA profile (the gene complex
responsible for encoding the MHC proteins in humans) can also be extra-
polated from the sequencing data. This increases the accuracy of this pre-
diction, especially for the most common HLA profiles with more data
available for building the prediction algorithm (Warren et al., 2012;
Zhang et al., 2011). Currently, the algorithms predicting binding to
MHC class I molecules are the most accurate and commonly used. Several
bioinformatic platforms are already available. Among the first to be deve-
loped, we find SYFPEITHI (Rammensee et al., 1999), Rankpep (Reche
et al., 2002), and BIMAS (Parker et al., 1994). NetMHC (Lundegaard
et al., 2008) is a more recent and well-validated prediction tool based
on what is known as “artificial neural network.” This network is con-
stantly updated with real data, thus improving its accuracy, especially
for the most common alleles. Other platforms (SMM and SMMPMBEC)
are able to minimize the negative impact of experimental error and/or

poor data (Peters and Sette, 2005).
Some retrospective studies have demonstrated the reliability of these
bioinformatic tools. Fritsch and colleagues used the NetMHC algorithm
to confirm previously reported ex vivo data regarding neoantigens identi-
fied by CD8+ T cells. Most neoantigens (87%) were correctly predicted
to be strong binders (Fritsch et al., 2014). A second study van Buuren
et al. (2014) were able to correctly predict 70% of the neoantigens based
on exome sequencing data. Although these results are quite encouraging,
there are some important limitations. First, these prediction tools, in
general, do not work as well for rare HLA alleles; second, they have not
been optimized for MHC class II molecules; and third, the variability
in proteasome processing is very high and can be influenced by several
mechanisms. Thus, these tools’ sensitivity is not yet ready for clinical use
(Tenzer et al., 2005).
Notwithstanding, some recent studies, both in animal models and in pre-
clinical trials, illustrate the effectiveness of neoantigens prediction and
targeting in cancer therapy. Yadav et al. (2014) synthesized peptides rep-
resenting point mutations that generated MHCI-bound epitopes and which
were identified in the MC38 mouse colon adenocarcinoma cells. These
peptides, once injected in wild-type mice, were able to provoke tumor
growth inhibition upon stimulation of CD8+ T cells response. Similar
results were obtained in an analogous study, in which, upon whole exome
sequencing of the B16F10 murine melanoma, 50 peptides were generated.
Upon their injection in mice models, they were observed to stimulate
immune response and inhibit tumor development (Castle et al., 2012). Fol-
lowing the same vaccine-based strategy, a few preliminary preclinical trials
have started using melanoma as main tumor model. In one of them, the
exomes of the tumor of three patients were sequenced, and data were ana-
lyzed with a prediction algorithm that allowed identifying epitopes used to
stimulate autologous dendritic cells (Patrick-Ott, 2015; Washington
University School of Medicine, 2016). Once reinjected into the patient,
these cells could enhance neoantigen-specific T cells, although there were
no evident signs of clinical benefits (Mittendorf et al., 2012). In another one,
immune responses to vaccination with peptide identified by whole exome
sequencing and prioritized by MHC-binding predictions will be monitored
over time (Carreno et al., 2015). In BC, vaccine-based therapeutic strategies
have already been used with good results (Yoon et al., 2012). Given that
immune response in breast tumors is specific and important, its functional
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man, Bill Smith, the father of the children, for Mr. Covey himself had
locked the two up together every night, thus inviting the result.
But I will pursue this revolting subject no farther. No better
illustration of the unchaste, demoralizing, and debasing character of
slavery can be found, than is furnished in the fact that this
professedly Christian slaveholder, amidst all his prayers and hymns,
was shamelessly and boastfully encouraging and actually
compelling, in his own house, undisguised and unmitigated
fornication, as a means of increasing his stock. It was the system of
slavery which made this allowable, and which condemned the
slaveholder for buying a slave woman and devoting her to this life,
no more than for buying a cow and raising stock from her, and the
same rules were observed, with a view to increasing the number and
quality of the one, as of the other.
If at any one time in my life, more than another, I was made to
drink the bitterest dregs of slavery, that time was during the first six
months of my stay with this man Covey. We were worked all
weathers. It was never too hot, or too cold; it could never rain, blow,
snow, or hail too hard for us to work in the field. Work, work, work,
was scarcely more the order of the day than of the night. The longest
days were too short for him, and the shortest nights were too long for
him. I was somewhat unmanageable at the first, but a few months of
this discipline tamed me. Mr. Covey succeeded in breaking me—in
body, soul, and spirit. My natural elasticity was crushed; my intellect
languished; the disposition to read departed, the cheerful spark that
lingered about my eye died out; the dark night of slavery closed in
upon me, and behold a man transformed to a brute!
Sunday was my only leisure time. I spent this in a sort of beast-
like stupor, between sleeping and waking, under some large tree. At
times I would rise up, a flash of energetic freedom would dart
through my soul, accompanied with a faint beam of hope that
flickered for a moment, and then vanished. I sank down again,
mourning over my wretched condition. I was sometimes tempted to
take my life and that of Covey, but was prevented by a combination
of hope and fear. My sufferings, as I remember them now, seem like
a dream rather than a stern reality.
Our house stood within a few rods of the Chesapeake bay,
whose broad bosom was ever white with sails from every quarter of
the habitable globe. Those beautiful vessels, robed in white, and so
delightful to the eyes of freemen, were to me so many shrouded
ghosts, to terrify and torment me with thoughts of my wretched
condition. I have often, in the deep stillness of a summer’s Sabbath,
stood all alone upon the banks of that noble bay, and traced, with
saddened heart and tearful eye, the countless number of sails
moving off to the mighty ocean. The sight of these always affected
me powerfully. My thoughts would compel utterance; and there, with
no audience but the Almighty, I would pour out my soul’s complaint in
my rude way with an apostrophe to the moving multitude of ships.
“You are loosed from your moorings, and free. I am fast in my
chains, and am a slave! You move merrily before the gentle gale,
and I sadly before the bloody whip. You are freedom’s swift-winged
angels, that fly around the world; I am confined in bonds of iron. O,
that I were free! O, that I were on one of your gallant decks, and
under your protecting wing! Alas! betwixt me and you the turbid
waters roll. Go on, go on; O, that I could also go! Could I but swim! If
I could fly! O, why was I born a man, of whom to make a brute! The
glad ship is gone: she hides in the dim distance. I am left in the hell
of unending slavery. O, God, save me! God, deliver me! Let me be
free!—Is there any God? Why am I a slave? I will run away. I will not
stand it. Get caught or get clear, I’ll try it. I had as well die with ague
as with fever. I have only one life to lose. I had as well be killed
running as die standing. Only think of it: one hundred miles north,
and I am free! Try it? Yes! God helping me, I will. It cannot be that I
shall live and die a slave. I will take to the water. This very bay shall
yet bear me into freedom. The steamboats steer in a northeast
course from North Point; I will do the same; and when I get to the
head of the bay, I will turn my canoe adrift, and walk straight through
Delaware into Pennsylvania. When I get there I shall not be required
to have a pass: I will travel there without being disturbed. Let but the
first opportunity offer, and come what will, I am off. Meanwhile I will
try to bear the yoke. I am not the only slave in the world. Why should
I fret? I can bear as much as any of them. Besides I am but a boy
yet, and all boys are bound out to some one. It may be that my
misery in slavery will only increase my happiness when I get free.
There is a better day coming.”
I shall never be able to narrate half the mental experience
through which it was my lot to pass, during my stay at Covey’s. I was
completely wrecked, changed, and bewildered; goaded almost to
madness at one time, and at another, reconciling myself to my
wretched condition. All the kindness I had received at Baltimore, all
my former hopes and aspirations for usefulness in the world, and
even the happy moments spent in the exercises of religion,
contrasted with my then present lot, served but to increase my
anguish.
I suffered bodily as well as mentally. I had neither sufficient time
in which to eat, or to sleep, except on Sundays. The over-work, and
the brutal chastisements of which I was the victim, combined with
that ever-gnawing and soul-devouring thought—“I am a slave—a
slave for life—a slave with no rational ground to hope for freedom”—
rendered me a living embodiment of mental and physical
wretchedness.
CHAPTER XVI.
ANOTHER PRESSURE OF THE TYRANT’S
VICE.
Experience at Covey’s summed up—First six months severer than the

remaining six—Preliminaries to the change—Reasons for narrating the
circumstances—Scene in the treading-yard—Author taken ill—Escapes to
St. Michaels—The pursuit—Suffering in the woods—Talk with Master
Thomas—His beating—Driven back to Covey’s—The slaves never sick—
Natural to expect them to feign sickness—Laziness of slaveholders.
THE reader has but to repeat, in his mind, once a week the scene in
the woods, where Covey subjected me to his merciless lash, to have
a true idea of my bitter experience, during the first six months of the
breaking process through which he carried me. I have no heart to
repeat each separate transaction. Such a narration would fill a
volume much larger than the present one. I aim only to give the
reader a truthful impression of my slave-life, without unnecessarily
affecting him with harrowing details.
As I have intimated that my hardships were much greater during
the first six months of my stay at Covey’s than during the remainder
of the year, and as the change in my condition was owing to causes
which may help the reader to a better understanding of human
nature, when subjected to the terrible extremities of slavery, I will
narrate the circumstances of this change, although I may seem
thereby to applaud my own courage.
You have, dear reader, seen me humbled, degraded, broken
down, enslaved, and brutalized; and you understand how it was
done; now let us see the converse of all this, and how it was brought
about; and this will take us through the year 1834.
On one of the hottest days of the month of August, of the year
just mentioned, had the reader been passing through Covey’s farm,
he might have seen me at work in what was called the “treading-
yard”—a yard upon which wheat was trodden out from the straw by
the horses’ feet. I was there at work feeding the “fan,” or rather
bringing wheat to the fan, while Bill Smith was feeding. Our force
consisted of Bill Hughes, Bill Smith, and a slave by the name of Eli,
the latter having been hired for the occasion. The work was simple,
and required strength and activity, rather than any skill or
intelligence; and yet to one entirely unused to such work, it came
very hard. The heat was intense and overpowering, and there was
much hurry to get the wheat trodden out that day, through the fan;
since if that work was done an hour before sundown, the hands
would have, according to a promise of Covey, that hour added to
their night’s rest. I was not behind any of them in the wish to
complete the day’s work before sundown, and hence I struggled with
all my might to get it forward. The promise of one hour’s repose on a
week day was sufficient to quicken my pace, and to spur me on to
extra endeavor. Besides, we had all planned to go fishing, and I
certainly wished to have a hand in that. But I was disappointed, and
the day turned out to be one of the bitterest I ever experienced.
About three o’clock, while the sun was pouring down his burning
rays, and not a breeze was stirring, I broke down; my strength failed
me; I was seized with a violent aching of the head, attended with
extreme dizziness, and trembling in every limb. Finding what was
coming, and feeling it would never do to stop work, I nerved myself
up, and staggered on, until I fell by the side of the wheat fan, with a
feeling that the earth had fallen in upon me. This brought the entire
work to a dead stand. There was work for four: each one had his part
to perform, and each part depended on the other, so that when one
stopped, all were compelled to stop. Covey, who had become my
dread, was at the house, about a hundred yards from where I was
fanning, and instantly, upon hearing the fan stop, he came down to
the treading-yard to inquire into the cause of the stopping. Bill Smith
told him I was sick, and that I was unable longer to bring wheat to
the fan.
I had by this time crawled away under the side of a post-and-rail
fence in the shade, and was exceedingly ill. The intense heat of the
sun, the heavy dust rising from the fan, the stooping to take up the
wheat from the yard, together with the hurrying to get through, had
caused a rush of blood to my head. In this condition Covey, finding
out where I was, came to me; and after standing over me a while he
asked what the matter was. I told him as well as I could, for it was
with difficulty that I could speak. He gave me a savage kick in the
side which jarred my whole frame, and commanded me to get up.
The monster had obtained complete control over me, and if he had
commanded me to do any possible thing I should, in my then state of
mind, have endeavored to comply. I made an effort to rise, but fell
back in the attempt before gaining my feet. He gave me another
heavy kick, and again told me to rise. I again tried, and succeeded in
standing up; but upon stooping to get the tub with which I was
feeding the fan I again staggered and fell to the ground; and I must
have so fallen had I been sure that a hundred bullets would have
pierced me through as the consequence. While down in this sad
condition, and perfectly helpless, the merciless negro-breaker took
up the hickory slab with which Hughes had been striking off the
wheat to a level with the sides of the half-bushel measure (a very
hard weapon), and with the edge of it he dealt me a heavy blow on
my head which made a large gash, and caused the blood to run
freely, saying at the same time, “If you have got the headache I’ll
cure you.” This done, he ordered me again to rise, but I made no
effort to do so, for I had now made up my mind that it was useless,
and that the heartless villain might do his worst, he could but kill me
and that might put me out of my misery. Finding me unable to rise, or
rather despairing of my doing so, Covey left me, with a view to
getting on with the work without me. I was bleeding very freely, and
my face was soon covered with my warm blood. Cruel and merciless
as was the motive that dealt that blow, the wound was a fortunate
one for me. Bleeding was never more efficacious. The pain in my
head speedily abated, and I was soon able to rise. Covey had, as I
have said, left me to my fate, and the question was, shall I return to
my work, or shall I find my way to St. Michaels and make Capt. Auld
acquainted with the atrocious cruelty of his brother Covey and
beseech him to get me another master? Remembering the object he
had in view in placing me under the management of Covey, and
further, his cruel treatment of my poor crippled cousin Henny, and his
meanness in the matter of feeding and clothing his slaves, there was
little ground to hope for a favorable reception at the hands of Capt.
Thomas Auld. Nevertheless, I resolved to go straight to him, thinking
that, if not animated by motives of humanity, he might be induced to
interfere on my behalf from selfish considerations. “He cannot,” I
thought, “allow his property to be thus bruised and battered, marred
and defaced, and I will go to him about the matter.” In order to get to
St. Michaels by the most favorable and direct road I must walk seven
miles, and this, in my sad condition, was no easy performance. I had
already lost much blood, I was exhausted by over-exertion, my sides
were sore from the heavy blows planted there by the stout boots of
Mr. Covey, and I was in every way in an unfavorable plight for the
journey. I however watched my chance while the cruel and cunning
Covey was looking in an opposite direction, and started off across
the field for St. Michaels. This was a daring step. If it failed it would
only exasperate Covey, and increase the rigors of my bondage
during the remainder of my term of service under him; but the step
was taken, and I must go forward. I succeeded in getting nearly half
way across the broad field toward the woods, when Covey observed
me. I was still bleeding, and the exertion of running had started the
blood afresh. “Come back! Come back!” he vociferated, with threats
of what he would do if I did not return instantly. But disregarding his
calls and threats, I pressed on toward the woods as fast as my
feeble state would allow. Seeing no signs of my stopping he caused
his horse to be brought out and saddled, as if he intended to pursue
me. The race was now to be an unequal one, and thinking I might be
overhauled by him if I kept the main road I walked nearly the whole
distance in the woods, keeping far enough from the road to avoid
detection and pursuit. But I had not gone far before my little strength
again failed me, and I was obliged to lie down. The blood was still
oozing from the wound in my head, and for a time I suffered more
than I can describe. There I was in the deep woods, sick and
emaciated, pursued by a wretch whose character for revolting cruelty
beggars all opprobrious speech, bleeding and almost bloodless. I
was not without the fear of bleeding to death. The thought of dying in
the woods all alone, and of being torn in pieces by the buzzards, had
not yet been rendered tolerable by my many troubles and hardships,
and I was glad when the shade of the trees and the cool evening
breeze combined with my matted hair to stop the flow of blood. After
lying there about three-quarters of an hour brooding over the
singular and mournful lot to which I was doomed, my mind passing
over the whole scale or circle of belief and unbelief, from faith in the
over-ruling Providence of God, to the blackest atheism, I again took
up my journey toward St. Michaels, more weary and sad than on the
morning when I left Thomas Auld’s for the home of Covey. I was
bare-footed, bare-headed, and in my shirt sleeves. The way was
through briers and bogs, and I tore my feet often during the journey. I
was full five hours in going the seven or eight miles; partly because
of the difficulties of the way, and partly because of the feebleness
induced by my illness, bruises, and loss of blood.
On gaining my master’s store, I presented an appearance of
wretchedness and woe calculated to move any but a heart of stone.
From the crown of my head to the sole of my feet, there were marks
of blood. My hair was all clotted with dust and blood, and the back of
my shirt was literally stiff with the same. Briers and thorns had
scarred and torn my feet and legs. Had I escaped from a den of
tigers, I could not have looked worse. In this plight I appeared before
my professedly Christian master, humbly to invoke the interposition
of his power and authority, to protect me from further abuse and
violence. During the latter part of my tedious journey, I had begun to
hope that my master would now show himself in a nobler light than I
had before seen him. But I was disappointed. I had jumped from a
sinking ship into the sea; I had fled from a tiger to something worse. I
told him all the circumstances, as well as I could: how I was
endeavoring to please Covey; how hard I was at work in the present
instance; how unwillingly I sank down under the heat, toil, and pain;
the brutal manner in which Covey had kicked me in the side, the
gash cut in my head; my hesitation about troubling him (Capt. Auld)
with complaints; but that now I felt it would not be best longer to
conceal from him the outrages committed on me from time to time.
At first Master Thomas seemed somewhat affected by the story of
my wrongs, but he soon repressed whatever feeling he may have
had, and became as cold and hard as iron. It was impossible, at first,
as I stood before him, to seem indifferent. I distinctly saw his human
nature asserting its conviction against the slave system, which made
cases like mine possible; but, as I have said, humanity fell before the
systematic tyranny of slavery. He first walked the floor, apparently
much agitated by my story, and the spectacle I presented; but soon it
was his turn to talk. He began moderately by finding excuses for
Covey, and ended with a full justification of him, and a passionate
condemnation of me. He had no doubt I deserved the flogging. He
did not believe I was sick; I was only endeavoring to get rid of work.
My dizziness was laziness, and Covey did right to flog me as he had
done. After thus fairly annihilating me, and arousing himself by his
eloquence, he fiercely demanded what I wished him to do in the
case! With such a knockdown to all my hopes, and feeling as I did
my entire subjection to his power, I had very little heart to reply. I
must not assert my innocence of the allegations he had piled up
against me, for that would be impudence. The guilt of a slave was
always and everywhere presumed, and the innocence of the
slaveholder, or employer, was always asserted. The word of the
slave against this presumption was generally treated as impudence,
worthy of punishment. “Do you dare to contradict me, you rascal?”
was a final silencer of counter-statements from the lips of a slave.
Calming down a little, in view of my silence and hesitation, and
perhaps a little touched at my forlorn and miserable appearance, he
inquired again, what I wanted him to do? Thus invited a second time,
I told him I wished him to allow me to get a new home, and to find a
new master; that as sure as I went back to live again with Mr. Covey,
I should be killed by him; that he would never forgive my coming
home with complaints; that since I had lived with him he had almost
crushed my spirit, and I believed he would ruin me for future service,
and that my life was not safe in his hands. This Master Thomas (my
brother in the church) regarded as “nonsense.” There was no danger
that Mr. Covey would kill me; he was a good man, industrious and
religious; and he would not think of removing me from that home;
“besides,” said he—and this I found was the most distressing
thought of all to him—“if you should leave Covey now that your year
is but half expired, I should lose your wages for the entire year. You
belong to Mr. Covey for one year, and you must go back to him,
come what will; and you must not trouble me with any more stories;
and if you don’t go immediately home, I’ll get hold of you myself.”
This was just what I expected when I found he had prejudged the
case against me. “But, sir,” I said, “I am sick and tired, and I cannot
get home to-night.” At this he somewhat relented, and finally allowed
me to stay the night, but said I must be off early in the morning, and
concluded his directions by making me swallow a huge dose of
Epsom salts, which was about the only medicine ever administered
to slaves.
It was quite natural for Master Thomas to presume I was feigning
sickness to escape work, for he probably thought that were he in the
place of a slave, with no wages for his work, no praise for well-doing,
no motive for toil but the lash, he would try every possible scheme by
which to escape labor. I say I have no doubt of this; the reason is,
that there were not, under the whole heavens, a set of men who
cultivated such a dread of labor as did the slaveholders. The charge
of laziness against the slaves was ever on their lips, and was the
standing apology for every species of cruelty and brutality. These
men did indeed literally “bind heavy burdens, grievous to be borne,
and laid them upon men’s shoulders, but they themselves would not
move them with one of their fingers.”
CHAPTER XVII.
THE LAST FLOGGING.
A sleepless night—Return to Covey’s—Punished by him—The chase

defeated—Vengeance postponed—Musings in the woods—The
alternative—Deplorable spectacle—Night in the woods—Expected attack
—Accosted by Sandy—A friend, not a master—Sandy’s hospitality—The
ash-cake supper—Interview with Sandy—His advice—Sandy a conjurer
as well as a Christian—The magic root—Strange meeting with Covey—
His manner—Covey’s Sunday face—Author’s defensive resolve—The
fight—The victory, and its results.
SLEEP does not always come to the relief of the weary in body, and
broken in spirit; especially is it so when past troubles only
foreshadow coming disasters. My last hope had been extinguished.
My master, who I did not venture to hope would protect me as a
man, had now refused to protect me as his property, and had cast
me back, covered with reproaches and bruises, into the hands of
one who was a stranger to that mercy which is the soul of the
religion he professed. May the reader never know what it is to spend
such a night as was that to me, which heralded my return to the den
of horrors from which I had made a temporary escape.
I remained—sleep I did not—all night at St. Michaels, and in the
morning (Saturday) I started off, obedient to the order of Master
Thomas, feeling that I had no friend on earth, and doubting if I had
one in heaven. I reached Covey’s about nine o’clock; and just as I
stepped into the field, before I had reached the house, true to his
snakish habits, Covey darted out at me from a fence corner, in which
he had secreted himself for the purpose of securing me. He was
provided with a cowskin and a rope, and he evidently intended to tie
me up, and wreak his vengeance on me to the fullest extent. I should
have been an easy prey had he succeeded in getting his hands upon
me, for I had taken no refreshment since noon on Friday; and this,
with the other trying circumstances, had greatly reduced my
strength. I, however, darted back into the woods before the ferocious
hound could reach me, and buried myself in a thicket, where he lost
sight of me. The cornfield afforded me shelter in getting to the
woods. But for the tall corn, Covey would have overtaken me, and
made me his captive. He was much chagrined that he did not, and
gave up the chase very reluctantly, as I could see by his angry
movements, as he returned to the house.
Well, now I am clear of Covey and his lash, for a little time. I am
in the wood, buried in its somber gloom, and hushed in its solemn
silence; hidden from all human eyes, shut in with nature, and with
nature’s God, and absent from all human contrivances. Here was a
good place to pray; to pray for help, for deliverance—a prayer I had
often made before. But how could I pray? Covey could pray—Capt.
Auld could pray. I would fain pray; but doubts arising, partly from my
neglect of the means of grace, and partly from the sham religion
which everywhere prevailed, cast in my mind a doubt upon all
religion, and led me to the conviction that prayers were unavailing
and delusive.
Life in itself had almost become burdensome to me. All my
outward relations were against me; I must stay here and starve, or
go home to Covey’s and have my flesh torn to pieces and my spirit
humbled under the cruel lash of Covey. These were the alternatives
before me. The day was long and irksome. I was weak from the toils
of the previous day, and from want of food and sleep, and I had been
so little concerned about my appearance that I had not yet washed
the blood from my garments. I was an object of horror, even to
myself. Life in Baltimore, when most oppressive, was a paradise to
this. What had I done, what had my parents done, that such a life as
this should be mine? That day, in the woods, I would have
exchanged my manhood for the brutehood of an ox.
Night came. I was still in the woods, and still unresolved what to
do. Hunger had not yet pinched me to the point of going home, and I
laid myself down in the leaves to rest; for I had been watching for
hunters all day, but not being molested by them during the day, I
expected no disturbance from them during the night. I had come to
the conclusion that Covey relied upon hunger to drive me home, and
in this I was quite correct, for he made no effort to catch me after the
morning.
During the night I heard the step of a man in the woods. He was
coming toward the place where I lay. A person lying still has the
advantage over one walking in the woods in the day-time, and this
advantage is much greater at night. I was not able to engage in a
physical struggle, and I had recourse to the common resort of the
weak. I hid myself in the leaves to prevent discovery. But as the night
rambler in the woods drew nearer I found him to be a friend, not an
enemy, a slave of Mr. William Groomes of Easton, a kind-hearted
fellow named “Sandy.” Sandy lived with Mr. Kemp that year, about
four miles from St. Michaels. He, like myself, had been hired out that
year, but unlike myself had not been hired out to be broken. He was
the husband of a free woman who lived in the lower part of “Poppie
Neck,” and he was now on his way through the woods to see her
and to spend the Sabbath with her.
Found in the Woods by Sandy.
As soon as I had ascertained that the disturber of my solitude
was not an enemy, but the good-hearted Sandy—a man as famous
among the slaves of the neighborhood for his good nature as for his
good sense—I came out from my hiding-place and made myself
known to him. I explained the circumstances of the past two days
which had driven me to the woods, and he deeply compassionated
my distress. It was a bold thing for him to shelter me, and I could not
ask him to do so, for had I been found in his hut he would have
suffered the penalty of thirty-nine lashes on his bare back, if not
something worse. But Sandy was too generous to permit the fear of
punishment to prevent his relieving a brother bondman from hunger
and exposure, and therefore, on his own motion, I accompanied him
home to his wife—for the house and lot were hers, as she was a free
woman. It was about midnight, but his wife was called up, a fire was
made, some Indian meal was soon mixed with salt and water, and an
ash-cake was baked in a hurry, to relieve my hunger. Sandy’s wife
was not behind him in kindness; both seemed to esteem it a privilege
to succor me, for although I was hated by Covey and by my master I
was loved by the colored people, because they thought I was hated
for my knowledge, and persecuted because I was feared. I was the
only slave in that region who could read or write. There had been
one other man, belonging to Mr. Hugh Hamilton, who could read, but
he, poor fellow, had shortly after coming into the neighborhood been
sold off to the far south. I saw him ironed, in the cart, to be carried to
Easton for sale, pinioned like a yearling for the slaughter. My
knowledge was now the pride of my brother slaves, and no doubt
Sandy felt something of the general interest in me on that account.
The supper was soon ready, and though I have since feasted with
honorables, lord mayors, and aldermen over the sea, my supper on
ash-cake and cold water, with Sandy, was the meal of all my life
most sweet to my taste, and now most vivid to my memory.
Supper over, Sandy and I went into a discussion of what was
possible for me, under the perils and hardships which overshadowed
my path. The question was, must I go back to Covey, or must I
attempt to run away? Upon a careful survey the latter was found to
be impossible; for I was on a narrow neck of land, every avenue from
which would bring me in sight of pursuers. There was Chesapeake
Bay to the right, and “Pot-pie” river to the left, and St. Michaels and
its neighborhood occupied the only space through which there was
any retreat.
I found Sandy an old adviser. He was not only a religious man,
but he professed to believe in a system for which I have no name.
He was a genuine African, and had inherited some of the so-called
magical powers said to be possessed by the eastern nations. He told
me that he could help me; that in those very woods there was an
herb which in the morning might be found, possessing all the powers
required for my protection (I put his words in my own language), and
that if I would take his advice he would procure me the root of the
herb of which he spoke. He told me, further, that if I would take that
root and wear it on my right side it would be impossible for Covey to
strike me a blow; that with this root about my person no white man
could whip me. He said he had carried it for years, and that he had
fully tested its virtues. He had never received a blow from a
slaveholder since he carried it, and he never expected to receive
one, for he meant always to carry that root for protection. He knew
Covey well, for Mrs. Covey was the daughter of Mrs. Kemp; and he
(Sandy) had heard of the barbarous treatment to which I had been
subjected, and he wanted to do something for me.
Now all this talk about the root was to me very absurd and
ridiculous, if not positively sinful. I at first rejected the idea that the
simple carrying a root on my right side (a root, by the way, over
which I walked every time I went into the woods) could possess any
such magic power as he ascribed to it, and I was, therefore, not
disposed to cumber my pocket with it. I had a positive aversion to all
pretenders to “divination.” It was beneath one of my intelligence to
countenance such dealings with the devil as this power implied. But
with all my learning—it was really precious little—Sandy was more
than a match for me. “My book-learning,” he said, “had not kept
Covey off me” (a powerful argument just then), and he entreated me,
with flashing eyes, to try this. If it did me no good it could do me no
harm, and it would cost me nothing any way. Sandy was so earnest
and so confident of the good qualities of this weed that, to please
him, I was induced to take it. He had been to me the good
Samaritan, and had, almost providentially, found me and helped me
when I could not help myself; how did I know but that the hand of the
Lord was in it? With thoughts of this sort I took the roots from Sandy
and put them in my right-hand pocket.
This was of course Sunday morning. Sandy now urged me to go
home with all speed, and to walk up bravely to the house, as though
nothing had happened. I saw in Sandy too deep an insight into
human nature, with all his superstition, not to have some respect for
his advice; and perhaps, too, a slight gleam or shadow of his
superstition had fallen on me. At any rate, I started off toward
Covey’s as directed. Having, the previous night, poured my griefs
into Sandy’s ears and enlisted him in my behalf, having made his
wife a sharer in my sorrows, and having also become well refreshed
by sleep and food, I moved off quite courageously toward the
dreaded Covey’s. Singularly enough, just as I entered the yard gate I
met him and his wife, dressed in their Sunday best, looking as
smiling as angels, on their way to church. His manner perfectly
astonished me. There was something really benignant in his
countenance. He spoke to me as never before, told me that the pigs
had got into the lot and he wished me to go to drive them out;
inquired how I was, and seemed an altered man. This extraordinary
conduct really made me begin to think that Sandy’s herb had more
virtue in it than I, in my pride, had been willing to allow, and had the
day been other than Sunday I should have attributed Covey’s altered
manner solely to the power of the root. I suspected, however, that
the Sabbath, not the root, was the real explanation of the change.
His religion hindered him from breaking the Sabbath, but not from
breaking my skin on any other day than Sunday. He had more
respect for the day than for the man for whom the day was mercifully
given; for while he would cut and slash my body during the week, he
would on Sunday teach me the value of my soul, and the way of life
and salvation by Jesus Christ.
All went well with me till Monday morning; and then, whether the
root had lost its virtue, or whether my tormentor had gone deeper
into the black art than I had (as was sometimes said of him), or
whether he had obtained a special indulgence for his faithful
Sunday’s worship, it is not necessary for me to know or to inform the
reader; but this much I may say, the pious and benignant smile
which graced the face of Covey on Sunday wholly disappeared on
Monday.
Long before daylight I was called up to go feed, rub, and curry
the horses. I obeyed the call, as I should have done had it been
made at an earlier hour, for I had brought my mind to a firm resolve
during that Sunday’s reflection to obey every order, however
unreasonable, if it were possible, and if Mr. Covey should then
undertake to beat me to defend and protect myself to the best of my
ability. My religious views on the subject of resisting my master had
suffered a serious shock by the savage persecution to which I had
been subjected, and my hands were no longer tied by my religion.
Master Thomas’ indifference had severed the last link. I had back-
slidden from this point in the slaves’ religious creed, and I soon had
occasion to make my fallen state known to my Sunday-pious brother,
Covey.
While I was obeying his order to feed and get the horses ready
for the field, and when I was in the act of going up the stable loft, for
the purpose of throwing down some blades, Covey sneaked into the
stable, in his peculiar way, and seizing me suddenly by the leg, he
brought me to the stable-floor, giving my newly-mended body a
terrible jar. I now forgot all about my roots, and remembered my
pledge to stand up in my own defense. The brute was skilfully
endeavoring to get a slip-knot on my legs, before I could draw up my
feet. As soon as I found what he was up to, I gave a sudden spring
(my two days’ rest had been of much service to me) and by that
means, no doubt, he was able to bring me to the floor so heavily. He
was defeated in his plan of tying me. While down, he seemed to
think he had me very securely in his power. He little thought he was
—as the rowdies say—“in” for a “rough and tumble” fight: but such
was the fact. Whence came the daring spirit necessary to grapple
with a man, who eight-and-forty hours before, could, with his
slightest word, have made me tremble like a leaf in a storm, I do not
know; at any rate I was resolved to fight, and what was better still, I
actually was hard at it. The fighting madness had come upon me,
and I found my strong fingers firmly attached to the throat of the
tyrant, as heedless of consequences, at the moment, as if we stood
as equals before the law. The very color of the man was forgotten. I
felt supple as a cat, and was ready for him at every turn. Every blow
of his was parried, though I dealt no blows in return. I was strictly on
the defensive, preventing him from injuring me, rather than trying to
injure him. I flung him on the ground several times when he meant to
have hurled me there. I held him so firmly by the throat that his blood
followed my nails. He held me, and I held him.
All was fair thus far, and the contest was about equal. My
resistance was entirely unexpected, and Covey was taken all aback
by it, and he trembled in every limb. “Are you going to resist, you
scoundrel?” said he. To which I returned a polite “yes, sir,” steadily
gazing my interrogator in the eye, to meet the first approach or
dawning of the blow which I expected my answer would call forth.
But the conflict did not long remain equal. Covey soon cried lustily
for help; not that I was obtaining any marked advantage over him, or
was injuring him, but because he was gaining none over me, and
was not able, single-handed, to conquer me. He called for his cousin
Hughes to come to his assistance, and now the scene was changed.
I was compelled to give blows, as well as to parry them, and since I
was in any case to suffer for resistance, I felt (as the musty proverb
goes) that I “might as well be hanged for an old sheep as a lamb.” I
was still defensive toward Covey, but aggressive toward Hughes, on
whom at his first approach, I dealt a blow which fairly sickened him.
He went off, bending over with pain, and manifesting no disposition
to come again within my reach. The poor fellow was in the act of
trying to catch and tie my right hand, and while flattering himself with
success, I gave him the kick which sent him staggering away in pain,
at the same time that I held Covey with a firm hand.
Taken completely by surprise, Covey seemed to have lost his
usual strength and coolness. He was frightened, and stood puffing
and blowing, seemingly unable to command words or blows. When
he saw that Hughes was standing half bent with pain, his courage
quite gone, the cowardly tyrant asked if I “meant to persist in my
resistance.” I told him I “did mean to resist, come what might; that I
had been treated like a brute during the last six months, and that I
should stand it no longer.” With that he gave me a shake, and
attempted to drag me toward a stick of wood that was lying just
outside the stable door. He meant to knock me down with it, but just
as he leaned over to get the stick, I seized him with both hands, by
the collar, and with a vigorous and sudden snatch, I brought my
assailant harmlessly, his full length, on the not over clean ground, for
we were now in the cow-yard. He had selected the place for the
fight, and it was but right that he should have all the advantages of
his own selection.
By this time Bill, the hired man, came home. He had been to Mr.
Helmsley’s to spend Sunday with his nominal wife. Covey and I had
been at skirmishing from before daybreak till now, and the sun was
now shooting his beams almost over the eastern woods, and we
were still at it. I could not see where the matter was to terminate. He
evidently was afraid to let me go, lest I should again make off to the
woods, otherwise he would probably have obtained arms from the
house to frighten me. Holding me, he called upon Bill to assist him.
The scene here had something comic about it. Bill, who knew
precisely what Covey wished him to do, affected ignorance, and
pretended he did not know what to do. “What shall I do, Master
Covey?” said Bill. “Take hold of him!—take hold of him!” said Covey.
With a toss of his head, peculiar to Bill, he said, “indeed Master
Covey, I want to go to work.” “This is your work,” said Covey; “take
hold of him.” Bill replied, with spirit: “My master hired me here to
work, and not to help you whip Frederick.” It was my turn to speak.
“Bill,” said I, “don’t put your hands on me.” To which he replied: “My
God, Frederick, I ain’t goin’ to tech ye;” and Bill walked off leaving
Covey and myself to settle our differences as best we might.
But my present advantage was threatened when I saw Caroline
(the slave woman of Covey) coming to the cow-yard to milk, for she
was a powerful woman, and could have mastered me easily,
exhausted as I was.
As soon as she came near, Covey attempted to rally her to his
aid. Strangely, and fortunately, Caroline was in no humor to take a
hand in any such sport. We were all in open rebellion that morning.
Caroline answered the command of her master “to take hold of me,”
precisely as Bill had done, but in her it was at far greater peril, for
she was the slave of Covey, and he could do what he pleased with
her. It was not so with Bill, and Bill knew it. Samuel Harris, to whom
Bill belonged, did not allow his slaves to be beaten, unless they were

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International Review of Cell and

Molecular Biology Volume 331 Lorenzo

International Review of Cell and Molecular Biology

International Review of Cell and Molecular Biology

International Review of Cell and Molecular Biology

International Review of Cell and Molecular Biology 1st

International Review of Cell and Molecular Biology 1st

International Review of Cell and Molecular Biology 1st

International Review of Cell and Molecular Biology

International Review of Cell and Molecular Biology 1st

Editorial Advisory Board

First edition 2017

Copyright © 2017 Elsevier Inc. All rights reserved.

For information on all Academic Press publications

Publisher: Zoe Kruze

Breast Cancer Immunology

TICA (Spanish Group for

MBC metastatic breast cancer

eradication, or immune system evasion by the tumor (Becker et al., 2013).

2. MECHANISMS OF THE IMMUNE RESPONSE

MDSC induction is likely caused by a general inflammatory process in

(Buijs et al., 2011). Moreover, MDSCs modulate alternatively activated M1

and indirect inhibition through the secretion of antiinflammatory media-

of mediating Treg suppressive functions (Gavin et al., 2007; Zheng et al.,

3. IMMUNE SYSTEM IN THE THERAPEUTIC RESPONSE

The third signal of immunogenic cell death is the release of HMGB1, an

4. T-INFILTRATING LYMPHOCYTES MOLECULAR AND

In BC, as in many other tumor types, immunosurveillance is functionally

Finally, the working group’s guidelines underscore the importance of not

monocytes and VEGF, in patients undergoing neoadjuvant treatment with

reduced risk of metastasis in Basal-like, Luminal B, and HER2-enriched

clinical activity in HER2+ cancers has been explored by assessing clinical

5. NEOANTIGEN PREDICTION: GENOMIC TOOLS

extracted 20 distinct mutational signatures, some of them present in different

are able to minimize the negative impact of experimental error and/or

Experience at Covey’s summed up—First six months severer than the

A sleepless night—Return to Covey’s—Punished by him—The chase

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