Autoantibody Production in Cancer--The Humoral Immune Response toward Autologous Antigens in Cancer Patients

Abstract

A link between autoimmune responses and cancer via autoantibodies was first described in the 1950's. Since, autoantibodies have been studied for their potential use as cancer biomarkers, however the exact causes of their production remain to be elucidated. This review summarises current theories of the causes of autoantibody production in cancer namely: 1) defects in tolerance and inflammation, 2) changes in protein expression levels, 3) altered protein structure and 4) cellular death mechanisms. We also highlight the need for further research into this field to improve our understanding of autoantibodies as biomarkers for cancer development and progression.

Full text

Review
Autoantibody Production in Cancer—The Humoral Immune Response
toward Autologous Antigens in Cancer Patients
P. Zaenker
a,
⁎,E.S.Gray
a
,M.R.Ziman
a,b
a
School of Medical and Health Sciences, Edith Cowan University, Joondalup, Perth, WA, Australia
b
Department of Pathology and Laboratory Medicine, The University of Western Australia, Crawley, Perth, WA, Australia
abstractarticle info
Article history:
Received 18 January 2016
Accepted 23 January 2016
Available online xxxx
A link between autoimmune responses and cancer via autoantibodies was first described in the 1950s. Since, au-
toantibodies have been studied for their potential use as cancer biomarkers, however the exact causes of their
production remain to be elucidated. This review summarizes current theories of the causes of autoantibody pro-
duction in cancer, namely: 1) defects in tolerance and inflammation, 2) changes in protein expression levels, 3)
altered protein structure, and 4) cellular death mechanisms. We also highlight the need for further research into
this field to improve our understanding of autoantibodies as biomarkers for cancer development and
progression.
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
Keywords:
Autoantibody
Autoantibody production
Biomarker
Cancer
Immune surveillance
Humoral immune response
Contents
1. Introduction............................................................... 0
2. Tolerance defects and inflammation.................................................... 0
2.1. Tolerancedefects ......................................................... 0
2.2. DownregulationofregulatoryTcells ................................................ 0
2.3. Inflammation........................................................... 0
3. Changesinproteinexpressionlevels.................................................... 0
3.1. Overexpressionofthecorrespondingantigen ............................................ 0
3.2. Aberrantexpressionsiteofthecorrespondingantigen ........................................ 0
4. Alteredproteinstructure......................................................... 0
4.1. Neoepitopeexposure ....................................................... 0
4.2. Mutations ............................................................ 0
4.3. Post-translational modifications .................................................. 0
5. Celldeathmechanismscauseaberrantreleaseofintracellularantigens .................................... 0
6. Concludingremarksandfutureperspectives ................................................ 0
Conflictofinterest .............................................................. 0
Financialdisclosurestatement ......................................................... 0
Take-homemessages ............................................................. 0
References.................................................................. 0
Autoimmunity Reviews xxx (2016) xxx–xxx
Abbreviations: dsDNA,double-strandedDNA; MIF, macrophagemigration inhibitory factor;Ang-2, angiopoietin 2; CENPF,centromere protein F; Her2/neu, human epidermal growth
factor receptor 2; MUC1, mucin 1; IMP2, insulin-like growth factor mRNA-binding family member 2; AORF, alternative open reading frame; CTAG1B/NY-ESO-1, cancer testis antigen 1B;
OGFr, opioid growth factor receptor; PSA, prostate-specific antigen; TNF, tumor necrosis factor; MAGEA3, melanoma antigen A3; PASD1, cancer antigen containing the PAS domain 1;
TGFβ, transforminggrowth factor beta; NKG2D, natural killer group 2 member D; ERp5, disulphide isomerase; MICA, MHC class 1 chain-relatedprotein A; CTLA4, cytotoxic T lymphocyte
associated protein 4; ATP, adenosine triphosphate; HMGB1, high mobility group B box protein 1.
⁎Corresponding author at: Edith Cowan University, 270 Joondalup Drive, Joondalup, Perth, WA 6027, Australia. Tel.: +61 8 63045716.
E-mail address: p.zaenker@ecu. edu.au (P. Zaenker).
AUTREV-01816; No of Pages 7
http://dx.doi.org/10.1016/j.autrev.2016.01.017
1568-9972/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Contents lists available at ScienceDirect
Autoimmunity Reviews
journal homepage: www.elsevier.com/locate/autrev
Please cite this article as: Zaenker P, et al, Autoantibody Production in Cancer—The Humoral Immune Response toward Autologous Antigens in
Cancer Patients, Autoimmun Rev (2016), http://dx.doi.org/10.1016/j.autrev.2016.01.017

1. Introduction
The production of autoantibodies (AAbs) is believed to reflect great-
er immunologic reactivity in cancer patients and enhanced immune
surveillance for cancer cells [1]. Since tumors originate from autologous
cells containing self-antigens, it has been suggested that it is the abnor-
mal exposure or presentation of these antigens that facilitates an auto-
immune response [2].
Over the lastfew decades, AAbs have become of particular interest as
cancer biomarkers as they can be easily extracted from serum via min-
imally invasive blood collection. Moreover, they exhibit increasedlevels
in very early cancer stages [3] and are observed in patients with several
carcinomas, including breast [4], lung [5], gastrointestinal [3], ovarian
[6], and prostate [7]. What is more, their production may precede clini-
cal confirmation of a tumor by several months or years [8].Notably,one
of the first historical reports of anti-tumor protein p53 (p53) antibodies
indicated that the AAbs were detectable as early as 17–47 months prior
to clinical tumor manifestation in uranium workers at high risk of lung
cancer development [9]. Detection of AAbs has also been reported
during the transition to malignancy [10]. Furthermore, AAbs may be
valuable biomarkers as they are stable serological proteins [11] with
high levels in serum despite low levels of the corresponding antigen
[12]. Additionally, they persist for extended periods after the corre-
sponding antigen is no longer detectable [6], at lasting concentrations
and with long half-lives in blood, due to limited proteolysis and clear-
ance from the circulation [13], making sample handling less arduous.
Studies have focused primarily on identifying AAbs as biomarkers
rather than investigating the underlying causes of their production.
However, the latter may reveal clues to the mechanisms involved ren-
dering autologous proteins immunogenic. Such studies could not only
lead to the development of novel biomarker assays, but also to the iden-
tification of novel therapeutic targets.
At present, the existence of a specific anti-tumor immune response,
referred to as “cancer immunome”, indicates that tumors express anti-
gens that are recognized as foreign by the host [11]. In the early stages
of carcinogenesis, this immune response is thought to occur as a result
of immune surveillance, the process by which the immune system
recognizes and destroys autologous cells that have become cancerous
[2,11]. In fact, histological examination of tumor affected tissues
revealed the presence of large populations of tissue resident and circu-
lating T and B cells that participate actively in immune surveillance [14].
As part of this surveillance, antigen presenting cells (APCs),
i.e., dendritic cells, B cells, and macrophages, engulf, lyse, and present
tumor-associated antigens (TAAs) on their cell surface for recognition
by CD4+ helper T cells. Interaction between the APC and T helper cell
triggers the APC release of cytokine and chemokine signals, resulting
in T cell activation and proliferation. B cells with high affinity for a spe-
cific TAA encounter the antigen, engulf, lyse, and also display it on their
cell surface for recognition and binding by activated T helper cells [15].
Lymphocyte recirculation into secondary lymphoid organs and periph-
eral tissue sites enhances this process, maximizing the frequency of
transformed cell TAAs encountering naïve B cells. The binding ofactivat-
ed T cells to B cell displayed TAAs initiates further release of cytokines
and chemokines leading to B cell proliferation. A vast number of B lym-
phocytes primed against the same antigen are produced, some of which
will serve as memory cells and others as effector cells that differentiate
into antibody producing plasma cells responsible for the systemic re-
lease of the appropriate antibody [16].Antibody–TAA binding thus rep-
resents the end stage of the humoral mechanism capable of initiating
the destruction of transformed cells containing the corresponding anti-
gen by, for example, labeling them (via opsonization) for faster macro-
phage recognition and phagocytosis. Direct binding of antibodies to the
antigen can also block receptors associated with tumor cell proliferation
and survival and AAbs can drive antigen uptake via dendritic cell Fc
gamma receptors, leading to antigen cross-presentation and vigorous
CD4+ and CD8+ T cell responses, complement dependent cytotoxicity,
and natural killer cell-mediated antibody-dependent cellular cytotoxic-
ity [17].
It is interesting to note that prolonged inflammation and the subse-
quent tissue destruction associated with autoimmune diseases [18]
share many parallels with the humoral immune response to TAAs
[19]. In fact, a repertoire of autoantibodies is shared by autoimmune
conditions and cancer [20]. For example, 30% of all cancer patients
have circulating anti-nuclear antibodies (ANAs) in their sera [21],auto-
antibodies associated with Sjögren's syndrome, systemic sclerosis, and
systemic lupus erythematosus (SLE), while these are generally absent
or present at very low levels in healthy individuals [22].
The exact factors that contribute to an enhancement or disturbance
of immune surveillance leading to the production of autoantibodies in
cancer are however still illusive, and the question remains as to how
and why cellular components may be rendered immunogenic in cancer.
Here we summarize some of the major theories surrounding the pro-
duction of autoantibodies in cancer (Fig. 1), including loss of tolerance,
inflammation, and changes in antigen expression, as well as their
altered exposure or altered presentation, reduced degradation, post-
translational modifications (PTMs), and their aberrant location or
altered structure.
2. Tolerance defects and inflammation
2.1. Tolerance defects
Approximately half of the lymphocyte population present in gener-
ative lymphoid organs is capable of binding to autoantigens [20].In
order to eliminate self-reactive lymphocytes entering the general circu-
lation, all immature lymphocytes must undergo a series of checkpoints
with processes aimed at maintaining central tolerance (tolerance to
self). Lymphocytes will only mature successfully if they are non-
reactive to autologous antigens and possess functional polypeptide
chains necessary to build a functional pre-antigen receptor, pre-BCR,
and pre-TCR for B and T cells, respectively. Self-reactive lymphocytes
are either eliminated, by negative selection via clonal deletion facilitated
apoptosis [23] or converted into a non-reactive state of clonal anergy
[24]. Alternatively, they may be preserved by positive selection,
Fig. 1. Proposed causes of autoantibody production in cancer.
2P. Zaenker et al. / Autoimmunity Reviews xxx (2016) xxx–xxx
Please cite this article as: Zaenker P, et al, Autoantibody Production in Cancer—The Humoral Immune Response toward Autologous Antigens in
Cancer Patients, Autoimmun Rev (2016), http://dx.doi.org/10.1016/j.autrev.2016.01.017

provided their antigen receptor alteration is induced, also known as re-
ceptor editing or revision [25]. Self-reactive B cells that have escaped
primary developmental checkpoints are further controlled by additional
peripheral checkpoints such as germinal center arrest [26]. However,
the processes of maintaining central tolerance are complex and subject
to error. For example, maintenance of clonal anergy is problematic as it
requires constant receptor occupancy and signalling, and is easily re-
versed by dissociation of the corresponding self-antigen, resulting in
anergic self-reactive B cells regaining responsiveness and potentially
leading to the production of autoantibodies [27]. It is also believed
that inappropriate survival of auto reactive lymphocytes by escape
from clonal deletion results when their corresponding antigen is
expressed at levels not high enough to induce clonal deletion [15,16].
Additionally, autoantibodies generated against autologous nuclear
antigens are frequently found in cancer patient sera. Nuclear antigens
are structurally disordered and their intrinsic proteolytic instability
has been suggested to interfere with the binding efficiency to major his-
tocompatibility complex (MHC) class II receptors throughout the elim-
ination of self-reactive lymphocytes, enabling their escape from the
primary lymphoid organs. Exposure of some autologous nuclear anti-
gens to the immune system, i.e., following tumor cell lysis, may there-
fore result in the production of autoantibodies [28].
2.2. Downregulation of regulatory T cells
High titers of AAbs have been associated with regulatory T cell
(Treg) downregulation. In fact, delayed tumor growth due to a reduc-
tion of Tregs has been correlated with an increase in effector T helper
cells, germinal center B cells, and high titers of autoantibodies [29],
such as anti-double-stranded DNA (dsDNA), ANAs, macrophage migra-
tion inhibitory factor (MIF), and angiopoietin 2 (Ang-2) antibodies, in-
dicating a robust anti-tumor response in conjunction with cell-
mediated immune response mechanisms [30].
In a recent study, Alvarez Arias et al. [30] utilized Qa-1 (HLA-E in
humans) knock-in B6 Qa-1 D227 mice that harbor a point mutation in
the MHC class Ib molecule, capable of impairing binding of CD8+ Treg
subsets to the T cell receptor (TCR) leading to impairment of the Treg
suppressive activity. B6-DK mice were inoculated with B16 melanoma
cells engineered to express the “B16-OVA”ovalbumin transgene. In-
creased titers of anti-OVA antibodies and substantially delayed tumor
growth were observed in transgenic mice compared to wild-type con-
trol mice. Furthermore, passive transfer of antibodies from the treated
B6-DK mice into a new cohort of mice highlighted that these autoanti-
bodies could curtail tumor growth, with 60% of these mice remaining
tumor free. By contrast, mice treated with autoantibodies from B6
wild-type mice developed tumors over time [30]. Thus, the downregu-
lation of Tregs and an imbalanced CD8+ Treg/T helper cell ratio in favor
of the effector T helper cells appeared responsible for the increase in
protective autoantibody production (Fig. 2) in this model [30]. It may
be possible that the reduced production of Tregs or similar Treg
downregulatory mechanisms exist in human melanoma or other can-
cers, rendering Treg/TCR binding ineffective and promoting autoanti-
body production in some cancer patients, with potential beneficial
outcomes.
2.3. Inflammation
Autoimmune responses, such as the production of autoantibodies,
may be part of a chronic inflammatory response toward cancer cells
and are associated with an array of immunological pathways, including
the releaseof several cytokines discussed in later sections. Inflammation
may be maintained throughout the duration of the cancer and increases
the permeability of the nearby vasculature, thereby enabling easier ac-
cess of immune cells to the site of the malignancy [30].
An induction of inflammation in the tumor microenvironment has
been suggested to facilitate the release of intracellular antigens resultin g
in abnormal exposure of autologous antigens to the immune system,
which may provide an explanation for the vast number of autoanti-
bodies produced against intracellular antigens in cancer patients [28].
The tumor inflammatory microenvironment has therefore, along with
changes in expression patterns of corresponding antigens, been
regarded as one of the main causes of autoantibody production in
cancer patients [31].
Fig. 2. Downregulation of Tregs correlates with high titers of autoantibodies and delayed tumor growth.
3P. Zaenker et al. / Autoimmunity Reviews xxx (2016) xxx–xxx
Please cite this article as: Zaenker P, et al, Autoantibody Production in Cancer—The Humoral Immune Response toward Autologous Antigens in
Cancer Patients, Autoimmun Rev (2016), http://dx.doi.org/10.1016/j.autrev.2016.01.017

3. Changes in protein expression levels
3.1. Overexpression of the corresponding antigen
Most TAAs are non-mutated antigens thatare produced at low levels
in healthy cells and overexpressed during tumorigenesis, thus causing
immunogenicity presumably by presentation of their antigenic peptides
on human leukocyte antigen (HLA) class I molecules, at levels high
enough to exceed the engaged TCR threshold required for CD4+ T cell
activation, and thereby indirectly leading to antibody production
against these autologous antigens [32].
A recent study by Hong et al. [33] suggested that increased anti-cen-
tromere protein F (CENPF) autoantibody levels, detected in a cohort of
hepatocellular carcinoma patients, were likely produced in response
to the overexpression of the protein in these cancer cells. Similarly, a
strong correlation exists between human epidermal growth factor recep-
tor 2 (Her2/neu) overexpression, detectable in 30% of adenocarcinomas
[34], and the frequency of anti-Her2/neu antibodies found in breast can-
cer patients [35].Goodelletal.[35] reported no autoantibody occurrence
in breast cancer cases with low Her2/neu expression and 82% antibody
occurrence in cases with high target protein expression levels.
Mucin 1 (MUC1) is an integral membrane protein whose expression
is normally confined to the apical surface of breast ductal epithelium, a
site that is relatively isolated from the immune system. MUC1 overex-
pression is detectable in 90% of all adenocarcinomas and is associated
with increased aggressiveness of the tumor [36]. MUC1 autoantibodies,
commonly detected in breast adenocarcinoma patients, are thought to
be produced in response to overexpression of this target antigen [36].
Interestingly, the binding of antibodies to MUC1 is suggested to have
an inhibiting effect on tumor invasive functions [37].
In early-stage ovarian cancer, p53-specificantibodiesareproduced
due to the presence of mutant p53 in these tumors [38].Arecent
study by Anderson et al. [6], compared p53-specificautoantibodypro-
duction in patients with serous ovarian cancer to patients with non-
serous ovarian cancer type and found that in the latter cases, p53 anti-
bodies were still detectable but at much lower levels than in those
with serous ovarian cancer, consistent with the lower frequency of
p53 mutations in non-serous tumors [39]. Studies have demonstrated
that the half-life of mutant p53 is markedly increased to several hours
while the wild-type p53 displays a half-life of only a few minutes,
resulting in aberrant accumulation of mutated p53 in the cell nucleus.
Notably, immunogenic epitopes have been mapped primarily to both
the N- and C-terminal portions of p53, but not to the central portion
of the molecule which harbors the mutations, suggesting that the accu-
mulation of the protein rather than the mutations per se may result in
the generation of anti-p53 autoantibodies [40].
3.2. Aberrant expression site of the corresponding antigen
Autoantibody production in cancer patients may also be due to ex-
pression of the protein in an aberrant location.
Cancer testis (CT) expression is normally confined to distinct and
immune privileged locations within the body, such as the gametes of
the testis or ovaries and in trophoblasts of the placenta [41]. Their aber-
rant expression in somatic tissues has been associated with spontane-
ous autoantibody production in patients with various cancer types [42].
Similarly, autoantibody production against the oncofetal antigen
insulin-like growth factor mRNA-binding family member 2 (IMP2) has
been detected in the sera of 21% of hepatocellular carcinoma patients
but is not detectable in patients with precursor conditions such as
liver cirrhosis and chronic hepatitis. Oncofetal antigens are expressed
throughout prenatal development and cease expression in all tissues
shortly after birth, whereas their re-expression in adulthood has
been associated with malignant transformation. This abnormal re-
expression has been suggested to be the cause of autoantibody produc-
tion in hepatocellular carcinoma patients [43].
4. Altered protein structure
4.1. Neoepitope exposure
It has been observed that autoantibodies are largely raised against
intracellular self-antigens [28] that may be aberrantly expressed in can-
cer cells resulting in abnormal presentation of reactive neoepitopes to
theimmunesystem[44]. A neoepitope may be created by somatic mu-
tations that change the protein structure or an epitope normally located
within an unexposed region of the protein, may become exposed by a
conformational change or stereochemical alteration of the protein
structure, thus stimulating an immune response [45]. Furthermore, ge-
netic instability, a hallmark of carcinogenesis [46], results in expression
of neoantigens (containing neoepitopes) in tumor cells that in turn ini-
tiates an immune response against the tumor or the surrounding autol-
ogous tissue [47].
Certain structural motifs of antigens, including carbohydrate side
chains, multivalency, epitope repetition, highly charged cell surfaces
and coiled coils, have been found to enhance antigenicity [48]. Interest-
ingly, autoantibodies elicited as a result of modified proteins are often
able to bind to both the modified and unmodified form of the protein,
possibly due to the gradual expansion of the spectrum of specificities
recognized in B and/or T cell immune responses, also referred to as epi-
tope spreading [49].
Furthermore, naturally occurring and cancer-associated molecular
mimicry represented by the attachment of mimotopes, macromolecules
such as peptides that mimic the structure of an epitope onto an APC,
may also elicit a humoral immune response since the antibody for a
given epitope will equally recognize the mimotope [3].
4.2. Mutations
All cancers carry somatic mutations which vary in frequency de-
pending on thecancer type and its cause [50]. p53 is commonly mutated
in a variety of cancers [51]. Altered p53 proteins produced from mis-
sense mutations may have acquired neo-antigenic determinants that
stimulate antibody production [2,52]. Interestingly, despite the strong
autoantibody specificity toward mutated p53, only 20–40% of patients
with cancers harboring p53 missense mutations have p53 autoanti-
bodies in their sera, indicating that other causes, such as protein accu-
mulation as mentioned previously and HLA polymorphisms, must
exist for autoantibody production to be facilitated in patients [40].
Notably, while missense point mutations leading to altered protein
products are highly correlated with production of autoantibodies,
stop, splice, and frameshift mutations do not generally appear to cause
autoantibody production in lung cancer [53]. By contrast, immunologi-
cal responses to antigens altered by frameshift mutations, have been de-
tected in colorectal cancer patients [54].
Furthermore, the translation of mRNA from an alternative open
reading frame (AORF) can lead to the generation of proteins bearing im-
munogenic determinants that can trigger humoral immune responses
in cancer patients. For example, antigenic peptides encoded from the
AORF of cancer-testis antigen 1B (CTAG1B), also known as NY-ESO-1,
have been shown to elicit immunological responses in cancer patients
[55]. Moreover, antibodies to the opioid growth factor receptor (OGFr)
protein recognize an alternative-reading frame of the molecule in pa-
tients suffering from melanoma, prostate cancer, lung cancer, breast,
and ovarian cancer [56].
4.3. Post-translational modifications
Post-translational modifications of proteins such as glycosylation,
methylation, phosphorylation, sumoylation, citrillination, adenosine
diphosphate-ribosylation, ubiquitination, and acetylation [57] are
known to occur aberrantly in cancer, creating a vast diversity of modi-
fied proteins and greatly expanding the number of targets for cancer-
4P. Zaenker et al. / Autoimmunity Reviews xxx (2016) xxx–xxx
Please cite this article as: Zaenker P, et al, Autoantibody Production in Cancer—The Humoral Immune Response toward Autologous Antigens in
Cancer Patients, Autoimmun Rev (2016), http://dx.doi.org/10.1016/j.autrev.2016.01.017

specific autoantibody production [13]. In fact, over 140 unique amino
acid derivatives are produced as a result of post-translational modifica-
tions, which can be enzyme-mediated or spontaneous and can generate
a neoepitope or enhance self-epitope presentation, inducing an im-
mune response [44,57].
The most common cancer-related changes involve glycosylation
[58] and phosphorylation [59], which can affect antigen processing,
binding, and interaction of the MHC presented antigen with the TCR,
therefore activating the T helper response needed to mount a humoral
autoantibody response. Aberrant glycosylation, for example, has been
observed in many cancers, causing the presentation of modified epi-
topes as TAAs that can override tolerance and induce antibody produc-
tion in cancer patients. Tarp et al. [60] reported the presence of
immunodominant epitopes on glycosyl moieties of mucin 1, which in-
duce a humoral immuneresponse in mucin-transgenic mice.Antibodies
recognizing specific MUC1 O-glycopeptides have been detected in ovar-
ian, breast, and prostate cancer patients [61], but MUC1 glycopeptides
are also detectable at similarfrequencies in healthy cancer-free controls
and are therefore not cancer specific[62].
5. Cell death mechanisms cause aberrant release of intracellular
antigens
How exactly the processing and metabolism of proteins can lead to
the mounting of an autoimmune response in cancer patients has yet
to be determined. Some studies have suggested that the cleavage of
known nuclear and cytoplasmic proteins during apoptosis and/or accu-
mulation due to insufficient clearance of secondary necrotic cells may
play a role [39,63,64]. The ongoing destruction of tumor cells, through-
out tumor development and progression, is thought to arise as a result
of extensive cell proliferation and cell survival producing overwhelming
cellular stresses [63], including ongoing immunological attacks, altered
protein structures, genomic instability, DNA damage, altered transcrip-
tional networks, and signal transduction, hypoxia as well as deprivation
of nutrients [64]. Cell lysis following tumor cell necrosis and autophagy
results in thespillage of autologousintracellular tumor contents into the
blood, exposing cancer-specific, cancer-associated, or neoantigens to
the immune system and effectively triggering an immune response
and the production of autoantibodies [20,65,66]. For example, the re-
lease of prostate-specific antigen (PSA) into the serum of patients
with malignant prostate tumors arisesfrom cell lysis due to a disruption
in the tissues between the p rostate gland lumen and its c apillaries, lead-
ing to anti-PSA antibody production [67].
Tumor cell lysis may also be promoted through secretion of cyto-
kines, such as tumor necrosis factor (TNF) and interferon-γ,byThelper
cells. TNF is an agent that causes tumor cell death by inducing thrombo-
sis in tumorblood vessels, resulting in high levels of interferon-sensitive
tumors releasing large numbers of cellular antigens, thereby triggering
the production of multiple autoantibodies [68]. In a case study of one
patient with hepatocellular carcinoma following radiation therapy, in-
creases in TNFαwere observed [69]. In another case study of a melano-
ma patient, increased melanoma antigen A3 (MAGEA3) autoantibody
levels as well as autoantibodies against cancer antigen containing the
PAS domain 1 (PASD1) were reported to be present in the patients
sera following intracranial stereotactic radiosurgery and the com-
mencement of Ipilimumab treatment [70].Inbothcasestudies,the
authors observed additional clearance of non-irradiated tumors, better
known as the abscopal effect, after localized radiation therapy to the
main lesion. Both the abscopal effect and an observed rise in autoanti-
body levels was suggested to be due to tumor antigen release and
cytokine production upon cell death caused by irradiation of the
tumor [69,70].
Furthermore, macrophages, and dendritic cells release immune sup-
pressive mediators such as transforming growth factor beta (TGF-β)
and interleukin-10 (IL-10), following their capture of apoptotic cells,
thereby stimulating Tregs [71]. The capture of necrotic cells on the
other hand, activates effector immune responses, includingthe humoral
response, by initiating the release of pro-inflammatory cytokines such
as IL-1β, IL-6, IL-12, and IL-23, capable of attenuating the effects of
TGF-βand IL-10 [72]. Since cancer cells involve both apoptotic and ne-
crotic cell death concurrently, the immune system is faced with oppos-
ing signals of immune tolerance and immune effector responses, and as
a result, neither process gains full control.
The presentation ofTAAs on the surface of dyingtumor cells may be
responsible for directing the immune response toward an activated ef-
fector response state thereby promoting autoantibody production (Fig. 3).
Cells that have natural killer group 2 member D (NKG2D) ligands
presented on their cell surface, signal the presence of DNA damage
within the cell and thus become detectable by surveillance immune
cells [73]. In order to avoid recognition by the immune system, tumor
cells shed, mediated by disulphide isomerase (ERp5) [74], the MHC
class 1 chain-related protein A (MICA) protein leading to the downreg-
ulation of NKG2D ligands on the cell surface [75]. An interesting study
by Jinushi et al. [76] showed that patients on granulocyte macrophage
colony-stimulating factor-secreting tumor cell vaccines or cytotoxic T
lymphocyte associated protein 4 (CTLA-4) immunotherapy, generated
high titers of ERp5 and MICA antibodies and the production of these au-
toantibodies not only restored the immunologic cytotoxic attack against
tumor cellsdue to the maintenance of NKG2D ligands on the cancer cell
surface but also promoted cross-presentation of ingested tumor antigens
by ACPs, thereby further strengthening the immunological anti-tumor at-
tack. The release of additional danger signals such as adenosine triphos-
phate (ATP) and high mobility group B box protein 1 (HMGB1) from
cancer cells undergoing autophagy may further aid in tipping the scales
toward an effector immune response including AAb production [65].
Apoptotic cancer cells have also been found to present altered cleav-
age products and post-translationally modified self-proteins on surface
blebs which promote autoimmunity [77]. Additionally, regulatory de-
fects in apoptosis resulting in the maintenance of mutant/defective
cells may render these autologous cells immunogenic.
6. Concluding remarks and future perspectives
AAb levels are detectable many months prior to the clinical manifes-
tation of a tumor, persist for an extended period after removal of the
corresponding antigen bearing tumor and in many ways reflect the
overall immunogenicity and immune response toward the tumor.
To provide useful insights on the interplay between the humoral im-
mune response and cancer, future studies should seek to investigate the
causes of the production of AAb biomarkers. The exact mechanisms re-
sponsible for cancer-related autoantibody production are still largely
unknown. As reviewed here, to date, several theories have been pro-
posed which include 1) tolerance defects and inflammation, 2) changes
in expression levels, 3) altered protein structure, as well as 4) cellular
death mechanisms. It appears that it is the abnormal expression and/
or alteration in the structure of the corresponding antigen that is most
accountable for an immune response. However, failure of tolerance
mechanisms, inflammation, and cell death affect the context in which
the antigensare presented to the immunesystem, initiatingthe produc-
tion of AAbs, in concert with other immune responses against trans-
formed cancer cells. Due to the heterogeneity of cancer cells and the
varied genetic and epigenetic differences between individual cancer
patients, it is likely that anti-cancer humoral autoimmune responses
originate from an array of such causes.
Furthermore, whether an increase or decrease in AAbs is beneficial
to the overall patient survival is still controversial and seems to differ
with each AAb. For instance, increases in AAb levels may be associated
with complete tumor remission in some anti-cancer therapies [70].
While an increase of NY-ESO-1 autoantibodies has been shown to reflect
increases in tumor burden in several cancers [78], decreases in AAb levels
are observed with cancer progression in some cases. The cancer progres-
sion associated drop in AAb levels has been suggested to be due to “cancer
5P. Zaenker et al. / Autoimmunity Reviews xxx (2016) xxx–xxx
Please cite this article as: Zaenker P, et al, Autoantibody Production in Cancer—The Humoral Immune Response toward Autologous Antigens in
Cancer Patients, Autoimmun Rev (2016), http://dx.doi.org/10.1016/j.autrev.2016.01.017

immunoediting,”comprised of three phases, namely, elimination, equilib-
rium, and the escape phase, resulting in the survival of resistant, immune
tolerant metastatic cancer cells [79]. AAb levels may therefore aid in mon-
itoring the immune response when assessing the efficacy of existing and
novel therapeutic agents and may prove effective in disease stagi ng or as a
predictor of recurrences and a favorable clinical outcome.
Finally, the presence of some autoantibodies has been correlated
with indolent tumor growth and increased patient survival [80].There-
fore, understanding the causes of AAb production in different cancers
may lead to the development of novel therapeutic agents or the timely
application of existing treatments.
Conflict of interest
The authors of the manuscript declare no conflict of interest.
Financial disclosure statement
The author PZ receives ongoing financial support as part of an Inspir-
ing Minds scholarship at Edith Cowan University while the other authors
did not receive any additional financial support for this manuscript.
Take-home messages
•A repertoire of AAbs is shared by autoimmune disorders and cancer.
•The causes of cancer-related AAb production remain yet to be
elucidated.
•Tolerance defects, inflammation, and cell death affect TAA immune
presentation.
•Abnormal TAA expression and structure appear most accountable
for AAb production.
•AAbs may be useful diagnostic, prognostic, and surveillance cancer
biomarkers.
References
[1] Kaae J, Wohlfahrt J, Boyd HA, Wulf HC, Biggar RJ, Melbye M. The impact of autoim-
mune diseases on the incidence and prognosis of cutaneous malignant melanoma.
Cancer Epidemiol Biomarkers Prev 2007;16:1840–4.
[2] Pardoll D. Does the immune system see tumors as foreign or self? Annu Rev
Immunol 2003;21:807–39.
[3] Zayakin P, Ancāns G, Siliņa K, Meistere I, Kalņina Z, Andrejeva SD, et al. Tumor-
associated autoantibod y signat ure for th e early detection of gastric cancer . Int
J Cancer 2013;132:137–47.
[4] Anderson KS, Sibani S, Wallstrom G. Protein microarray signature of autoantibody
biomarkers for the early detection of breast cancer. J Proteome Res 2011;10:85–96.
[5] Chapman CJ, Thorpe AJ, Murray A. Immunobiomarkers in small cell lung cancer:
potential early cancer signals. Clin Cancer Res 2010;17:1474–80.
[6] Anderson KS, Cramer DW, Sibani S, Wallstrom G, Wong J, Park J, et al. Autoantibody
signature for the serologic detection of ovarian cancer. J Proteome Res 2015;14:
578–86.
[7] Wang X, Yu J, Sreekumar A, Varambally S, Shen R, Giacherio D. Autoantibody signa-
tures in p rostate cancer. N Engl J Med 2005;353:12 24–35.
[8] Caron M, Choquet-Kastylevsky G, Joubert-Caron R. Cancer immunomics: using auto-
antibody signatures for biomarker discovery. Mol Cell Proteomics 2007;6:1115–22.
[9] RoyahemJ, Conrad K, Frey M, Melhom J, FrankKH. Autoantibodies PredictiveParam-
eters of Tumor Development. Berlin: Pabst; 1988.
[10] Zhang JY, Tan EM. Autoantibodies to tumor-associated antigens as diagnostic bio-
markers in hepatocellular carci noma and other solid tumors. Expert Re v Mol
Diagn 2010;10:321–8.
[11] Anderson KS, LaBaer J. The sentinelwithin: exploiting the immunesystem for cancer
biomarkers. J Proteome Res 2005;4:1123–33.
[12] Lu H, Goodell V, Disis ML. Humoral immunity directed against tumor-associated an-
tigens as potential biomarkers for the early diag nosis of cancer. J Proteome Res
2008;7:1388–94.
[13] Pedersen JW, Wandall HH. Autoantibodies as biomarkers in cancer. Labmedicine
2011;42:623–8.
[14] Linnebacher M, Maletzki C. Tumor-infiltrating B cells. Oncoimmunology 2012;1:
1186–8.
[15] Goodnow CC, Sprent J, Fazekas de Groth B, Vinuesa CG. Cellular and genetic mecha-
nisms of self tolerance and autoimmunity. Nature 2005;435:590–7.
[16] Wardemann H, Nussenzweig MC. B-cell self-tolerance in humans. Adv Immunol
2007;95:83–110.
[17] Carter P. Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer
2001;1:118–29.
[18] Kamradt T, Mitchison NA. Tolerance and autoimmunity. N Engl J Med 2001;344:
655–64.
[19] Preiss S, Kammertoens T, Lambert C. Tumor-induced antibodies resemble the
response to tissue damage. Int J Cancer 2005;115:456–62.
[20] Bei R, Masuelli L, Palumbo C, Modesti M, ModestiA. A common repertoireof autoan-
tibodiesis shared by cancer andautoimmune disease patients: inflammation in their
induction and impact on tumor growth. Cancer Lett 2009;281:8–23.
[21] Torchilin VP, Lakoubov LZ, Estov Z. Antinuclear autoantibodies as potential antineo-
plastic agents. Trends Immunol 2001;22:424–7.
[22] Fernández-Madrid F, Karvonen RL, Ensley J, Kraut M, Granda JL, Alansari H, et al.
Spectra of antinuclear antibodies in patients with squamous cell carcinoma of the
lung and of the head and neck. Cancer Detect Prev 2005;29:59–65.
[23] Venanzi ES, Benoist C, Mathis D. Good riddance: thymocyte clonal deletion prevents
autoimmmunity. Curr Opin Immunol 2004;16:197–202.
[24] Gauld SB, Merrell KT, Cambier JC. Silencing autoreactive B cells by anergy: a fresh
perspective. Curr Opin Immunol 2006;18:292–7.
[25] Gay D, Saunders T, Camper S, Weigert M. Receptor editing: an approach by
autoreactive B cells to escape tolerance. J Exp Med 1993;177:999–1008.
[26] Paul E, Lutz J, Erikson J, Carroll MC. Germinal center checkpoints in B cell tolerance in
3H9 transgenic mice. Int Immunol 2004;16:377–84.
Fig. 3. The presentation of TAAs on the tumor cell surface directs the immune response toward autoantibody production.
6P. Zaenker et al. / Autoimmunity Reviews xxx (2016) xxx–xxx
Please cite this article as: Zaenker P, et al, Autoantibody Production in Cancer—The Humoral Immune Response toward Autologous Antigens in
Cancer Patients, Autoimmun Rev (2016), http://dx.doi.org/10.1016/j.autrev.2016.01.017

[27] Ding C, Yan J. Regulation of autoreactive B cells: checkpoints and activation. Arch
Immunol Ther Exp (Warsz) 2007;55:83–9.
[28] Carl PL,Temple BR, Cohen PL. Most nuclear systemicautoantigens are extremely dis-
ordered proteins: implications for the etiology of systemic autoimmunity. Arthritis
Res Ther 2005;7:1360–74.
[29] Kim HJ, Verbinnen B, Tang X, Lu L, Cantor H. Inhibition of follicular T helper cells by
CD8 + Treg is essential for self tolerance. Nature 2010;467:328–32.
[30] Alvarez Arias DA, Kim H-J, Zhou P, Holderried TAW, Wang X, Dranoff G, et al. En-
hanced murine antitumour immunity after genetic disruption of CD8 + regulatory
T-cell activity. Cancer Immunol Res 2014;2:207–16.
[31] Nanda NK, Sercarz EE. Induction of anti-self-immunity to scure cancer. Cell 1995;82:
13–7.
[32] Watanabe N, Arase H, Onodera M, Ohashi PS, Saito T. The quantity of TCR signal de-
termines positive selection and lineage commitment of T cells. J Immunol 2000;165:
6252–61.
[33] Hong Y, Zhang B, Jia JD, Huang J. Identification of autoantibody based serological
marker for early diagnosis of hepatocellular carcinoma by serological proteome
analysis combined with protein microarray. Hepatology 2013;58(Suppl. 1).
[34] Menard S, Casalini P, Campiglio M. Role of HER2/neu in tumor progression and therapy.
Cell Mol Life Sci 2004;61:2965–78.
[35] Goodell V, Waisman J, Salazar JG. Level of HER-2/neu protein expression in breast
cancer may affect the development of endogenous HER-2/neu-specific immunity.
Mol Cancer Ther 2008;7:449–54.
[36] Yonezawa S, Goto M, Yamanda N. Expression profiles of MUC1, MUC2, and MUC4
mucins in human neoplasms and theirrelationship withbiological behaviour. Prote-
omics 2008;8:3329–41.
[37] Agrawal B, Krantz MJ, Reddish MA. Cancer-associated MUC1 mucin inhibits human
T-cell proliferation, which is reversible by IL-2. Nat Med 1998;4:43–9.
[38] Goodell V, Salazar LG, Urban N, Drescher CW, Gray H, Swensen RE, et al. Antibody
immunity to the p53 oncogenic protein is a prognostic indicator in ovarian cancer.
J Clin Oncol 2006;24:762–8.
[39] Anderson KS, Wong J, Vitonis A, Crum CP, Sluss PM, LaBaer J, et al. p53 autoanti-
bodies as potential detection and prognostic biomarkers in serous ovarian Cancer.
cancer Epidemiol Biomarkers Prev 2010;19:859–68.
[40] Soussi T. p53 antibodies in the sera of patients with various types of cancer: a
review. Cancer Res 2000;60:1777–88.
[41] Scanlan MJ, Gure AO, Old LJ. Cancer/testis antigens: an expanding family of targets
for cancer immunotherapy. Immunol Rev 2002;188:22–32.
[42] Simpson AJG, Caballero OL, Jungbluth A. Cancer/testis antigens, gametogenesis and
cancer. Nat Rev Cancer 2005;5:615–26.
[43] Zhang JY, Chan EK, Peng XX, Tan EM.A novel cytoplasmic protein with RNA-binding
motifs is an autoantigen in human hepatocellular carcinoma. J Exp Med 1999;189:
1101–10.
[44] Hanash S. Harnessing immunity for cancer marker discovery. Nat Biotechnol 2003;
21:37–8.
[45] Genovese F, Karsdal MA, Leeming DJ, Liu T, Wang X. Molecular serum markers of
liver fibrosis. Biomark Insights 2012;105.
[46] Fearon ER, Vogelstein BA. Genetic model for colorectal tumorogenesis. Cell 1990;61:
759–67.
[47] Joseph CG, Darrah E, Shah AA, Skora AD, Casciola-Rosen LA, Wigley FM, et al.
Associa tio n of autoimmune disease scleroderma with an immunologic response
to cancer. Science 2014;343:152.
[48] Plotz PH. The autoantibody repertoire: searching for order. Nat Rev Immunol 2003;
3:73–8.
[49] James JA, Harley JB. B-cell epitope spreading in autoimmunity. Immunol Rev 1998;
164:185–200.
[50] Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SAJR, Behjati S, Biankin AV, et al.
Signatures of mutational processes in human cancer. Nature 2013;500:415–21.
[51] KandothC, McLellan MD, Vandin F, Ye K, Niu B, Lu C,et al. Mutational landscape and
significance across 12 major cancer types. Nature 2013;502:333–9.
[52] Winter SF, Minna JD, Johnson BE, Takahashi T,Gazdar AF, Carbone DP. Development
of antibodies against p53 in lung cancer patients appears to be dependent on the
type of p53 mutation. Cancer Res 1992;52:4168–74.
[53] Reuschenbach M, von Knebel Doeberitz M, Wentzensen N. A systematic review of
humoral immune responses against tumor antigens. Cancer Immunol Immunother
2009;58:1535–44.
[54] Schwitalle Y, Kloor M, Eiermann S, Linnebacher M, Kienle P, Knaebel HP, et al. Im-
mune response against frameshift-induced neopeptides in HNPCC patients and
healthy HNPCC mutation carriers. Gastroenterology 2008;134:988–97.
[55] Wang RF, Johnston SL, Zeng G, Topalian SL, Schwartzentruber DJ, Rosenberg SA. A
breast and melanoma-shared tumor antigen: T cell responses to anti-genic peptides
translated from different open reading frames. J Immunol 1998;161:3598–606.
[56] Mollick JA, FS Hodi, RJ Soiffer, LM Nadler, Dranoff G. MUC1-like tandem repeat
proteins are broadly immunogenic in cancer patients. Cancer Immun 2003;3:3.
[57] Anderton SM. Pos t-translational modifications of self-antigens: implications for
autoimmunity. Curr Opin Immunol 2004;16.
[58] Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle.
Proc Natl Acad Sci USA 2002;99:10231–3.
[59] Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature 2001;411.
[60] Tarp MA,Sorensen AL, Mandel U, Paulsen H, Burchell J, Taylor-PapadimitriouJ, et al.
Identification of a novel cancer-specific immunodominant glycopeptide epitope in
the MUC1 tandem repeat. Glycobiology 2007;17:197–209.
[61] Wandall HH, Blixt O, Tarp MA. Cancer biomarkers defined by autoantibody signa-
tures to abberrant O-glycopeptide epitopes. Cancer Res 2010;70:1306–13.
[62] Burford B, Genty-Maharaj A, Graham R. Autoantibodies to MUC1 glycopeptides
cannot be used as a screening assay for early detection of breast, ovarian, lung or
pancreatic cancer. Br J Cancer 2013;108:2045–55.
[63] Edinger AL, Thompson CB. Death by design: apoptosis, necrosis and autophagy. Curr
Opin Cell Biol 2004;16:663–9.
[64] Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med
2004;10:789–99.
[65] Peter C, Wesselborg S, Herrmann M, Lauber K. Dangerous attraction: phagocyte
recruitment and danger signals of apoptotic and necrotic cells. Apoptosis 2010;
15:1007–28.
[66] Tan EM, Zhang J. Autoantibodies to tumor-associated antigens: reporters from the
immune system. Immunol Rev 2008;222:328.
[67] Pan D, McCahy P. Patient knowledge about prostate-specific antigen (PSA) andpros-
tate cancer in Australia. Warragul, Victoria: Department of Urology, Casey Hospital,
Berwick and West Gippsland Health Service; 2011.
[68] de Visser KE, Eichten A, Coussens LM. Paradoxial roles of the immune system during
cancer development. Nat Rev Cancer 2006;6:24–37.
[69] Ohba K, Omagari K, Nakamura T. Abscopal regression of heatocellular carcinoma
after radiotherapy for bone metastasis. Gut 1998;43:575–7.
[70] Stamell EF, Wolchok JD, Gnjatic S, Lee NY, Brownell I. The abscopal effect associated
with a systemic anti-melanoma immune response. Int J Radiat Oncol Biol Phys 2013;
85:293–5.
[71] Jinushi M, Nakazaki Y, DouganM, Carrasco DR, Mihm M, Dranoff G. MFG-E8 mediat-
ed uptake of apoptotic cells by APCs links the pro- and anti-inflammatory activities
of GM-CSF. J Clin Invest 2007;117:1902–13.
[72] Rock KL, Kono H. The inflammatory response to cell death. Annu Rev Pathol 2007.
[73] GasserS, Orsulic S, Brown EJ, Raulet DH. The DNA damage pathway regulates innate
immune system ligands of the NKG2D receptor. Nature 2005;436:1186–90.
[74] Kaiser BK, Yim D, Chow IT. Disulph ide-isomerase-enabled shedding of tumour-
associated NKG2D ligands. Nature 2007;447:482–6.
[75] Groh V, Wu J, Yee C, Spies T. Tumour-derived soluble MIC ligands impair expession
of NKG2D and T-cell activation. Nature 2002;419:734–8.
[76] Jinushi M, Hodi FS, Dranoff G. Therapy-induced antibodies to MHC class 1 chain-
relat ed protein A antagonize immune suppression and stimulate antitumor cytotoxicity.
Proc Natl Acad Sci U S A 2006;103:9190–5.
[77] Doyle HA, Mamula MJ. Post-translational protein modifications in antigen recognition
and autoimmunity. Trends Immunol 2001;22:443–9.
[78] Barrow C, Browning J, MacGregor D. Tumor antigen expression in melanoma varies
according to antigen and stage. Clin Cancer Res 2006;12:764–71.
[79] Schreiber RD, Old LJ. Cancer immunoediting: integrating immunity's roles in cancer
suppression and promotion. Science 2011;331:1565–70.
[80] GrausF, Dalmau J, Rene R, ToraM, Malats N, Verschuuren JJ, et al. Anti-Hu antibodies
in patients with small-cell lung cancer: association with complete response to
therapy and improved survival. Clin Oncol 1997;15:2866–72.
7P. Zaenker et al. / Autoimmunity Reviews xxx (2016) xxx–xxx
Please cite this article as: Zaenker P, et al, Autoantibody Production in Cancer—The Humoral Immune Response toward Autologous Antigens in
Cancer Patients, Autoimmun Rev (2016), http://dx.doi.org/10.1016/j.autrev.2016.01.017