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33 Circulating Biomarkers inOncology: Areas ofApplication, Critical Issues, andPerspectives
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Biomarkers intheBlood andinTissues:
AComplementary Approach
This chapter deals with circulating biomarkers; however, it
may be helpful to briey examine some aspects related to the
measurement of biomarkers in the tumor tissue, in order to
identify the advantages and limits that the two matrices present and to better understand why the determination of biomarkers in tissues or in blood can answer different clinical
questions.
Tissue biomarkers are receiving prevalent interest because
of the need to identify molecular targets for new anticancer
drugs. The determination of biomarkers in tissues has several
points of strength. First, the biomarker is measured in the
target tissue. Thus, no biomarker dilution occurs (as it does
in the blood), and possible nonspecic or background signals
should be mainly related to analytical aspects and eventually
constrained. In the case of tissue biomarkers, diagnostic sensitivity and specicity are both associated with analytical
specicity and sensitivity; in other words, the ability to correctly identify the molecule of interest is mainly dependent
on the characteristics of the method of determination. The
problem of tissue heterogeneity can also be addressed by
several approaches, such as, for example, by selecting areas
or cell types of interest by laser-assisted microdissection.
Interpretation of biomarker expression at the tissue level thus
appears relatively straightforward.
However, the determination of biomarkers in a tissue has
some signicant limitations associated with biological, conceptual, and technical aspects, which must be considered in
both clinical application and research design.
The biological aspects concern: (1) the intrinsic complexity of the molecular mechanisms; (2) the complex interaction
of tumor cells with the microenvironment; and (3) the redundancy of the regulatory mechanisms of the tumor cell. These
variables are associated with the inherent biological complexity of development and progression of cancer and explain
the difculties in identifying appropriate biomarkers as drug
targets for some signaling pathways. However, it can reasonably be expected that the increase of knowledge of cancer
cell biology and the progressive development of technologies will lead to an increasingly wide variety of measurable
biomarkers as well as to a progressive improvement of diagnostic sensitivity and specicity of the available methods.
The conceptual limitations of the determination of a biomarker in the tissue matrix are twofold: (1) the representativeness of the sample and (2) the timing of the sampling in
relation to the possible variability of biomarker expression
over time. Regarding representativeness, biomarker determination is performed with a minimal amount of tissue as only
a few milligrams of the tumor tissue are usually examined by
immunohistochemistry under routine conditions. Thus, it is
difcult to consider the immunohistochemical determination
of a biomarker as a truly quantitative measure in terms of
representativeness of the examined tissue specimen. This
issue might be of poor relevance in homogeneous tumors,
but it should be considered in the case of intrinsically heterogeneous malignancies. The second conceptual aspect concerns the one-shot feature of the measure, which shows the
expression of the biomarker in a single site and at a single
time point. Therefore, clinical decisions during the disease
course are based on the presence/absence of the biomarker
measured in a sample examined even years earlier, rather
than on the current phenotype of the malignancy. This entails
a risk of error in the characterization of the disease and, consequently, in therapeutic decisions. For example, HER2
expression may differ in metastases and in primary breast
cancer in approximately 25% of cases; nonetheless, the therapy of patients with metastatic breast cancer is based on
HER2 determination obtained in the primary tumor; a comparable degree of variability between the primary tumor and
metastases has been reported for estrogen receptors.
Finally, a technical issue must be considered. Biomarkers
are measured in tissues mainly by molecular biology techniques or immunohistochemistry. The latter is extensively
used for routine biomarker measurements for clinical
decision- making. Immunohistochemical techniques are typically qualitative, and the assumption that biomarker determination (e.g., estrogen receptors) is quantitative stems from
the fact that the result is often expressed as a quantitative
score or a percentage. However, it has been shown that the
routine immunohistochemical method performed on xed
tissue samples does not lead to accurate quantitative results,
even when performed in highly qualied laboratories.
The measurement of biomarkers in a tumor tissue is crucial for prognostic assessment and the prediction of response
to anticancer agents, but it is inherently unsuitable for risk
assessment, diagnosis, early recognition of recurrence, and
therapy monitoring. In these situations, biomarkers can only
be measured in blood. Table33.1 summarizes the scenarios
in which biomarkers can provide clinical information if measured in a tumor tissue or in blood.
Biomarkers in the blood can be determined at all stages of
the disease, can be easily repeated over time, and their measurement is typically quantitative and expressed on a continuous scale. The categorization into positive and negative
results based on a threshold value is a conventional dichotomization operation. However, different from tissue biomarkers, they are measured in a biological uid distant from the
malignancy, and, therefore, they are a surrogate measure of
the actual amount of biomarkers produced and released by
the tumor tissue. In fact, the amount of biomarker in the
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Table 33.1 Indications for biomarker use in different clinical scenarios according to the biological material: comparison between the tumor
tissue and peripheral blood
Indication for
biomarker use
Tumor
Clinical scenarios
Assessment of occupational or environmental
cancer risk
Screening of asymptomatic general population No Ye s
Surveillance of subjects with increased cancer
risk
Differential diagnosis in symptomatic patients No Ye s
Initial diagnostic workup (prognosis) Yes No/
Initial diagnostic workup (prediction of
response to therapy)
Long-term monitoring after curative treatment
of the primary tumor (early detection of
relapse)
Therapy monitoring in patients with advanced
or disseminated disease
tissue Blood
No Ye s
No Ye s
Yes
Yes No
No Ye s
No Ye s
blood results from several variables in addition to the production and release by the tumor tissue. Such variables
include: (1) the possible production of the biomarker by normal tissues; (2) the possible presence in the blood of substances that cross-react with the measurement system; (3)
the dilution in blood volume; and (4) metabolism and clearance. All of these factors may contribute to the blood concentration of a biomarker in a variable and unpredictable
manner, both between different patients and in the same
patient at different time points. Since the biomarker level in
the blood results from different factors that add up algebraically (some factors cause an increase, others a decrease), it
is necessary to use a threshold value to classify the biomarker
result as positive or negative. The threshold value is usually
determined from the distribution of the biomarker in a control group of healthy subjects or patients with non- oncological
diseases. The classication of the biomarker results with reference to a positive/negative threshold value in subjects with
the neoplasm of interest and in control subjects allows to
classify patients and controls into four groups: (1) true positives (subjects with the disease and positive biomarker); (2)
false negatives (subjects with the disease and negative biomarker); (3) true negatives (subjects without the disease and
negative biomarker); and (4) false positives (subjects without
the disease and positive biomarker).
The frequencies of patients and controls falling into the
four groups enable the calculation of the diagnostic performance of the biomarker, such as sensitivity, specicity, predictive value, and likelihood ratio, as summarized in
Table33.2.
The non-specicity of a malignancy and the relatively
low diagnostic sensitivity represent the commonly recog-
Table 33.2
Indicator Formula Denition
Sensitivity (SS) TP/(TP+FN) Proportion of subjects with the
Specicity (SP) TN/(TN+FP) Proportion of subjects without
Positive
predictive value
(PPV)
Negative
predictive value
(NPV)
Diagnostic
accuracy
Likelihood
ratio of a
positive result
(LR+)
Likelihood
ratio of a
negative result
(LR−)
FN false negatives, FP false positives, TN true negatives, TP true
positives
Measures of diagnostic accuracy
disease with a positive test
result in the total of subjects
with the disease
the disease with a negative test
result in the total of subjects
without the disease
TP/(TP+FP) Proportion of subjects with the
TN/(TN+FN) Proportion of subjects without
(TP+TN)/
(TP+FN+TN+FP)
TP/FP Proportion of subjects with the
FN/TN Proportion of subjects with the
disease with a positive test
result in the total of subjects
with positive results
the disease with a negative test
result in the total of subjects
with negative test results
Proportion of subjects correctly
classied by the test, in the
total of examined subjects
disease with a positive test
result divided by the proportion
of subjects without the disease
with a positive test result
disease with a negative test
result divided by the proportion
of subjects without the disease
with a negative test result
nized limitations of the vast majority of, if not all, circulating
biomarkers available for clinical use.
Poor specicity is an intrinsic problem of known biomarkers. The so-called “tumor markers” are biochemical
substances generally produced by several organs or cell
types. They are therefore not specic to a given tumor but to
the tissue or organ in which the tumor develops. The amount
of biomarker produced and/or released by the tissue cells is
low or very low in a physiological status but may increase
under pathological conditions. These certainly include the
development of a malignancy but also encompass several
other conditions, such as, for example, the increase of the
size of the organ (hyperplasia), inammatory events, infections, autoimmune diseases, traumas, surgery, and administration of several drugs. In addition, the impairment of liver
or kidney function can indirectly cause a spurious increase in
circulating levels of some biomarkers by reducing their
metabolism or clearance.
The poor diagnostic sensitivity is due to two major components: the frequently low concentration of the biomarker
in the blood and the analytical sensitivity of the method of
measurement. The low amount of biomarker present in the
blood in patients bearing small tumors is an objective issue,
which impairs the possible use of the biomarker in early
33 Circulating Biomarkers inOncology: Areas ofApplication, Critical Issues, andPerspectives
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diagnosis, and limits its value for the early detection of the
relapse during the follow-up. When the amount of tumor
tissue is still minimal, the circulating concentration of the
biomarker may remain within the normal range. The analysis of biomarker increments over time seems a promising
approach to improve the diagnostic efcacy for early cancer. The progressive development of the malignancy may
lead to a gradual increase of the biomarker concentration,
even when it is within the normal range. The variation of
biomarker levels between serial samples has been investigated in several clinical scenarios, such as early detection
of ovarian and prostate cancers or early recognition of
relapse in different types of malignancies, including breast,
ovarian, and colon cancers. The criteria to standardize the
interpretation of serial sample- based biomarker measurements have been proposed to facilitate the conduct of clinical trials, but the available results are in general still
inconclusive. This is due at least in part to the relatively low
analytical sensitivity of many assay methods. Indeed, most
commercially available methods for measuring “tumor
markers” are designed to offer linearity of dose–response
interpolation over a broad concentration range, ensuring
reliable results in a wide variety of clinical situations. This
choice, aimed at facilitating the routine use of biomarker
determination in clinical laboratories, entails the assumption that the quantitative level of the biomarker below the
threshold value– classied as negative– is clinically uninformative. The assay design often implies that analytical
precision is optimal for biomarker concentrations close to,
or above, the threshold value, with analytical variability
tending to worsen as the biomarker concentration decreases.
To be clinically meaningful, differences between serial
samples must be more signicant than the analytical and
biological variability of the biomarker. The poor reproducibility and comparability between the results reported in
different studies on biomarker variations over time may be
due, at least in part, to unsatisfactory assay precision at low
biomarker concentrations, which negatively affects both
analytical sensitivity and the calculation of biological variability. The availability of differently designed assay methods, targeted toward low biomarker concentrations and
therefore characterized by higher analytical sensitivity and
high precision in the low value range, would allow to better
explore the clinical use of decision criteria based on serial
sample variations.
In conclusion, determining biomarkers in the tumor tissue and in blood is not an alternative but a complementary
approach, presenting different advantages and limits. In
both matrices, signicant margins for improvement are
recognizable, related to the development of knowledge,
innovation of the study design, and improvement of technological aspects. Research policies in the eld of biomarkers should consider the value of diversities between
tissue and blood biomarkers, thus avoiding unnecessary
redundancies and overlaps and emphasizing the advantage of complementarities.
Circulating Biomarkers inClinical Practice
An increasing number of studies on candidate biomarkers for
clinical application are continuously published. However,
preliminary, often very promising results are rarely conrmed by further studies. Only for a few biomarkers evidence is sufcient to recommend their clinical use. These
biomarkers, identied 30–40 years ago, are the so-called
“tumor-associated antigens,” whose circulating levels correlate with tumor bulk.
An essential aspect in the choice of biomarkers to be used
in clinical practice concerns the types of literature to refer to.
Published studies can be schematically classied into three
categories with respect to the source of the information: (1)
primary research, which concerns the original results of experimental studies; (2) secondary research, consisting of metaanalyses and systematic reviews; and (3) tertiary research,
which leads to the production of guidelines. Primary studies
and most meta-analyses and systematic reviews mainly deal
with the efcacy of biomarkers; in other words, they assess
diagnostic performance (e.g., sensitivity, specicity, predictive
values, likelihood ratio) against a reference diagnostic criterion (usually, histological ndings) or in comparison with
other established biomarkers. The evaluation of efcacy is a
necessary, but not sufcient, condition for recommending the
clinical use of a biomarker, which requires an evaluation of the
impact of the biomarker on clinical outcomes or organizational processes taking into account other context variables;
for example, an effective biomarker should not be recommended if effective therapies to treat the diagnosed condition
are not available. Clinical practice guidelines, as dened by
the Institute of Medicine (IOM), are documents containing
recommendations to optimize patient care based on the systematic review of evidence and the assessment of the benets
and harms of alternative care options. Therefore, it should be
advisable to refer to guidelines when deciding whether to use
a biomarker in clinical decisions.
In order to review the state of the art on the use of circulating biomarkers in clinical practice, guidelines on solid
tumors produced worldwide from 2009 to 2015 have been
searched and reviewed and recommendations on circulating
biomarkers have been summarized. Information on biomarkers was extracted separately for 21 types of solid tumors and
summarized for each malignancy into the following clinical
scenarios: (1) screening of asymptomatic general population; (2) surveillance of individuals with increased cancer
risk; (3) differential diagnosis in symptomatic patients; (4)
initial diagnostic workup; (5) postoperative evaluation; (6)
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long-term monitoring after curative treatment of the primary
tumor; and (7) therapy monitoring in patients with advanced
or disseminated disease.
The principal indications on the use of biomarkers in different clinical scenarios are summarized below. Please refer
to the recommended lectures for detailed information.
Screening ofAsymptomatic General
Population
This scenario refers to the systematic search for the disease
in asymptomatic subjects through organized, general population screening programs. The goal is to reduce cancerspecic mortality through early detection of the disease. It is
offered by health-care organizations when evidence is sufcient to ensure that screening offers more advantages than
disadvantages or risks to the population. No biomarker is
currently recommended in this setting. In fact, the diagnostic
specicity and sensitivity of available biomarkers is not sufcient in relation to the prevalence of the cancers being
screened to ensure adequate predictive values. It should be
noted that available guidelines recommend against the use of
prostate-specic antigen (PSA) in organized prostate screening programs, based on the results of randomized trials of
consistent size. Guidelines have a similar position in ovarian
cancer, recommending against the use of CA125 to screen
asymptomatic women. Similar considerations are reported
by guidelines concerning the use of biomarkers to screen for
cancer asymptomatic people referring to the physician for a
checkup (i.e., spontaneous or opportunistic screening).
Surveillance ofIndividuals at Increased Risk
ethnicity, guidelines present controversial indications.
Finally, guidelines do not recommend regular CA125 monitoring for women with a hereditary–familial risk for ovarian
cancer.
Dierential Diagnosis inSymptomatic Patients
This scenario concerns the differential diagnosis between a
malignancy and other non-oncological conditions in subjects
with suspicious symptoms for cancer. In the differential
diagnosis, only a few biomarkers for a few malignancies are
indicated. This is the case of Ct in the clinical suspicion of
medullary thyroid cancer, PSA in the case of suspected prostate cancer, and CA125in women with a suspected adnexal
mass. Guideline recommendations are more controversial
for other malignancies, in which biomarkers are recommended by some guidelines and discouraged by others.
Examples are the use of AFP for space-occupying lesions of
the liver, of CA19.9in suspicious biliary tract or pancreatic
cancers, and of nuclear matrix protein 22 (NMP22) in suspicious bladder cancer. Finally, determining lactate dehydrogenase (LDH) in kidney cancer is recommended only when
metastatic dissemination of the disease is suspected.
Excluded from this scenario are patients presenting
generic symptoms (asthenia, weight loss, inappetence, anemia) or clinical situations commonly considered “paraneoplastic” (recurrent thrombophlebitis, neurological
symptoms); in these circumstances, biomarkers are not
recommended.
To be noted, all guidelines explicitly recommend that biomarkers are not used as a standalone diagnostic procedure,
but they must be considered in association with imaging
techniques only.
This scenario concerns the systematic search for cancer in
people with a higher risk of developing a malignancy than
the general population, due to familiarity, ethnicity, lifestyle,
or environmental conditions associated with increased cancer risk. In these subjects, the predictive value of biomarkers
may increase because the prevalence of malignancy is higher
than that in the general population. Guidelines advocate
using calcitonin (Ct) in the surveillance of relatives of
patients with hereditary medullary thyroid cancer. The position of the guidelines regarding the surveillance of subjects
with liver diseases associated with an increased risk of developing hepatocarcinoma is controversial; some guidelines
recommend using AFP, while others are against it. Likewise,
in the case of surveillance of patients with sclerosing cholangitis, carbohydrate antigen 19.9 (CA19.9) is recommended
by some guidelines, while others recommend not to use this
biomarker. Also, regarding the evaluation of PSA in subjects
with an increased risk of prostate cancer due to familiarity or
Initial Diagnostic Workup
In this scenario, biomarkers can be measured to have a baseline value before any treatment. In addition, biomarkers can
provide additional prognostic indications to help guide therapeutic decisions. Guidelines agree in recommending the
determination of: CEA in colorectal cancer; AFP in hepatocarcinoma; CA19.9in pancreatic cancer; CA125in endometrial cancer; PSA in prostate cancer; AFP, human chorionic
gonadotropin β-subunit (β-hCG), and LDH in testicular germ
cell tumors and non-epithelial ovarian tumors; LDH in melanoma and lung cancer; Ct in medullary thyroid cancer; thyroglobulin (Tg) and anti-thyroglobulin antibodies (AbTg) in
differentiated thyroid cancers; and LDH and C-reactive protein in mesothelioma. Biomarker results are included in
algorithms to dene risk classes in some malignancies, such
as prostate cancer and testicular germ cell tumors.
33 Circulating Biomarkers inOncology: Areas ofApplication, Critical Issues, andPerspectives
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Postoperative Evaluation
After the treatment with curative intent of the primary tumor,
the patient may be reevaluated to conrm the radicality in the
case of surgery or to estimate the probability of the treatment
success in the case of radiotherapy. In this scenario, guidelines recommend: AFP in hepatocarcinoma; CA19.9in pancreatic cancer; CA125 in epithelial ovarian cancer; AFP,
β-hCG, and LDH in germ cell tumors of the testis and nonepithelial ovarian cancer; PSA in prostate cancer; CEA in
non-small cell lung cancer; Epstein–Barr virus plasma DNA
(EBV-DNA) in nasopharyngeal cancers; Tg, AbTg, and
thyroid- stimulating hormone (TSH) in differentiated thyroid
cancers; and Ct and CEA in medullary thyroid cancers.
Long-Term Monitoring after Curative
Treatment ofthePrimary Tumor
Long-term surveillance (follow-up) is offered to patients
treated with curative intent because early recognition of the
relapse and a timely treatment should reduce mortality and/
or improve quality of life. Guidelines agree in recommending: CEA in colorectal cancer; AFP in hepatocellular carcinoma; AFP, β-hCG, and LDH in germ cell tumors of the
testis and non-epithelial tumors of the ovary; PSA in prostate
cancer; LDH in kidney cancer; EBV-DNA in nasopharyngeal cancers; Tg, AbTg, and TSH in differentiated thyroid
cancers; and Ct and CEA in medullary thyroid cancers.
Guidelines also agree not to recommend any biomarkers in
the follow-up of cervical cancer, bladder cancer, and melanoma. As concerns breast cancer and epithelial ovarian cancer, guidelines are against the use of biomarkers in the
follow-up of asymptomatic women and recommend their use
(carbohydrate antigen 15.3 (CA15.3) and CEA in breast cancer and CA125in ovarian cancer) only in symptomatic cases
or when clinically indicated. Finally, different guidelines
provide inconsistent recommendations in endometrial cancer, with some documents recommending against using
CA125, while others consider it as an optional test.
Therapy Monitoring inPatients withAdvanced
or Disseminated Disease
for the early detection of nonresponders or to recognize the
onset of resistance, in order to promptly modify the therapeutic schemes. In this scenario, the use of biomarkers is
relatively well established, and several guidelines advocate
their use. Guidelines recommend: CEA in colorectal cancer;
AFP in hepatocarcinoma; CA19.9 in pancreatic cancer;
CA19.9 and CEA in biliary tract tumors; CA15.3 and CEA
in breast cancer; LDH in melanoma; mesothelin (soluble
mesothelin-related peptide (SMRP)) in mesothelioma;
CA125in epithelial tumors of the ovary; CEA, a fragment of
cytokeratin 19 (Cyfra 21.1) and gastrin-releasing peptide
(GRP) precursor (pro-GRP) in lung malignancies; PSA in
prostate cancer; LDH in kidney cancer; AFP, β-hCG, and
LDH in germ cell tumors of the testis and non-epithelial
tumors of the ovary; TSH in differentiated thyroid cancers;
and Ct and CEA in medullary thyroid cancers.
From a general point of view, biomarkers can be grouped
into three categories with reference to the available
recommendations:
1. Biomarkers indicated in only one malignancy: PSA in
prostate cancer; Ct in medullary thyroid cancer; Tg,
AbTg, and TSH in differentiated thyroid cancers;
CA15.3in breast cancer; SMRP in mesothelioma; EBVDNA in nasopharyngeal cancer; and NMP22in bladder
cancer
2. Biomarkers indicated in a limited number of tumors char-
acterized by a common or similar histological type:
β-hCG in germ cell tumors of the testis and non-epithelial
tumors of the ovary
3. Biomarkers expressed by more than one neoplasm; rec-
ommendations for the cynical use of biomarkers in this
latter group are summarized in Table33.3.
Neuroendocrine tumors constitute a separate subject as
they encompass a very heterogeneous group of malignancies
that can express and release various molecules, some of
which are associated with specic symptoms. The number of
possible histological types and anatomical localization, and
the variety of biomarkers potentially measurable in relation
to tumor site and histological variants, do not allow for an
exhaustive summary here. Because of the relatively low incidence of these tumors, we prefer to direct readers to specic
literature.
This scenario encompasses locally advanced malignances
and distant metastases. In some cancers, such as differentiated thyroid cancers and germ cell tumors of the testis, therapies may impact the survival and probability of cure even in
the metastatic spread. In other malignancies, the therapeutic
intent is usually aimed mainly at symptom control and palliation of metastases. In both cases (probability of cure and
palliation), monitoring the response to the therapy is crucial
Measuring Methods
Biomarkers are routinely measured by immune assays,
which are based on the high-afnity binding of a specic
antibody to the target antigen. The majority of commercially
available immunoassays are fully standardized and available
on commercial platforms for routine use. Nevertheless, for
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Table 33.3 Biomarkers expressed by different tumor types: recommendations of clinical practice guidelines
Clinical scenario
Marker Malignancy Screening Diagnosis
AFP Hepatocarcinoma Yes
Germinal tumors of the testis No Ye s Ye s Ye s Ye s Ye s
Non-epithelial tumors of the
ovary
CA125 Endometrial cancer Ye s
Epithelial cancer of the ovary No Ye s
CA19.9 Colorectal cancer No No
Pancreatic cancer Yes
CEA Colorectal cancer Yes Yes
Breast cancer Yes
Lung cancer Yes
Medullary thyroid cancer Ye s Ye s Ye s Ye s Yes
LDH Melanoma Yes No Yes
Kidney tumors Ye s
Germinal tumors of the testis No Ye s Ye s Ye s Ye s Ye s
Non-epithelial tumors of the
ovary
AFP α-fetoprotein, CA19.9 carbohydrate antigen 19.9, CA125 carbohydrate antigen 125, CEA carcinoembryonic antigen, LDH lactate
dehydrogenase
a
In patients with chronic liver disease and an increased risk of developing hepatocellular carcinoma
b
Only in association with imaging techniques
c
In symptomatic patients or with other clinical indications
d
In the absence of symptoms or other clinical indications
e
In the absence of jaundice
f
In the suspicion of advanced disease
a
Yes
Yes
Initial
workup
b
Yes Yes
b
e
f
b
Yes Yes
Postoperative
evaluation
Long-term
follow-up
Yes
d
Yesc/No
c
d
/No
Yes Yes
M. Gion et al.
Therapy
monitoring
Yes
Yes
several biomarkers, international reference standards are not
available. Moreover, evidence shows that results obtained
with different immunoassays are not always interchangeable
even if calibrated using the same reference standard. The
advent of new immunoassays based on multiplexing technologies, capable of determining several biomarkers simultaneously, may further emphasize this issue. Therefore, from
an operational point of view, patients who are to be followed
over time should be advised to refer to the same laboratory
for the determination of biomarkers.
Beside the indications on the type of biomarker recommended for clinical purposes, guidelines also provide general information on the proper use of biomarkers, which are
summarized in Box 33.1.
Biomarkers Associated withBiological
Mechanisms
Several innovative biomarkers associated with known molecular mechanisms, such as inammation, angiogenesis,
matrix degradation, microenvironment interactions, or
deregulation of cellular signaling systems, have been intensively investigated.
The interest toward mechanism biomarkers is due to their
association with the biological characteristics and functions
of the malignancy. Several biomarkers in this category are
related to either malignancy aggressiveness or cellular mechanisms, which may be a target of specic anticancer agents.
Therefore, a solid biological rationale exists to assume that
these biomarkers may have a prognostic (relationship with
the aggressiveness of the neoplasm) or a predictive (relationship with the target of dedicated drugs) role. However, studies published so far on circulating levels of these biomarkers
have failed to provide conclusive data to support their use for
clinical purposes. This systematic failure of research on
mechanism biomarkers at the circulating level is quite surprising, especially when dealing with biomarkers that are
effective and useful in guiding therapeutic choices when
measured in the tissue. A paradigmatic example is HER2/
neu, a biomarker routinely measured in the breast cancer tissue to assess patients’ suitability for treatment with monoclonal antibodies targeted against HER2/neu (trastuzumab).
In contrast, the measurement of HER2/neu in blood has not
led to consistent results for a possible clinical use. Studies on
circulating levels of mechanism biomarkers deserve a general remark. In the majority of published studies, these biomarkers have been evaluated as classical “tumor markers.”
They have been examined with reference to the characteristics of the malignancy (e.g., histological type, disease stage)
or clinical goals (e.g., diagnosis, monitoring, prediction of
response to therapy). In fact, these biomarkers are linked to
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general pathophysiological mechanisms. Classical “tumor
markers” are also produced in small amounts by normal
cells, and their circulating levels may increase due to causes
other than cancer. However, classical “tumor markers” are
not the expression of well-dened pathophysiological mechanisms. Conversely, mechanism biomarkers are molecules
that actively participate in known and well-dened biological functions. This is the case of chemokines, factors regulating neoangiogenesis, or proteases and their inhibitors. These
biomarkers are expressed when a mechanism is activated,
regardless of the cause. For this reason, these molecules cannot be specic to any given malignancy. Therefore, to evaluate these biomarkers, it is necessary to consider different
approaches from those used so far for the classical “tumor
markers.” For instance, the pre-analytical phase should be
differently considered. Many of these biomarkers circulate
freely in blood, but they are also released from either blood
vessel cells or cellular elements of blood, i.e., leukocytes and
platelets. Blood represents a multi-compartmental matrix for
these biomarkers. In the case of vascular endothelial growth
factor (VEGF), it is well established that the concentration
measured in the serum does not correspond to the amount of
biomarker released by the tumor but also reects the released
biomarker by the cellular components of the blood. It has
been observed that the VEGF concentrations in the serum
may signicantly increase up to hundreds of times by extending the time between collection and centrifugation from 1 to
3h, a time interval compatible with ordinary specimen management and considered in many published studies as a
“standard procedure” of serum preparation. This aspect represents a limitation if the biomarker is considered a “tumor
marker,” but it may be instead a source of information on the
potential relationships of the host microenvironment with the
malignancy. As a matter of fact, the host’s production of
inammatory cytokines and neoangiogenic factors may be
evaluated as a regulatory element of cancer development and
progression. Therefore, mechanism biomarkers should be
studied as both a possible signal of the tumor cell and an
indicator of the host’s interaction with the malignancy. In
this perspective, research efforts should be focused on the
pre-analytical phase, evaluating the biomarkers in the plasma
obtained through different anticoagulants, serum collected
by standardized and reproducible procedures, and cellular
blood elements.
The second point of interest specic to mechanism biomarkers is their possible interactions in the blood with biological drugs targeted to the same biomarkers at the tissue
level. In the case of the administration of a monoclonal antibody directed against a biomarker expressed in the tumor
tissue, the concentration of the same biomarker in the blood
should not be considered a “neutral” element in the complex
drug/tissue biomarker/circulating biomarker interactions.
High circulating biomarker levels could bind the drug, thus
making it unavailable in adequate amounts to act on the
tumor tissue. From another point of view, drug-induced inhibition of the signaling system in the tumor tissue could
decrease the biomarker release in the blood in responder
patients. Conversely, an increased biomarker production and
release into the circulation could indicate the occurrence of
resistance. Considering the complexity of the possible interactions, the mere determination of a mechanism biomarker
as “tumor marker” is reductive, if not misleading. Some pilot
studies examined the relationships between circulating
marker levels and the response to biological drugs. An
inverse relationship between blood HER2/neu concentration
and response to trastuzumab has been described in women
with advanced breast cancer. Also, the importance of determining free VEGF in the plasma to assess the pharmacodynamics of bevacizumab has been shown, suggesting the
potential role of the biomarker in optimizing treatment and
tailoring doses and/or the duration of administration.
Recently, it has been found that bevacizumab induces a dosedependent blockade of VEGF internalization, demonstrating
that the increase in VEGF during bevacizumab administration does not represent a mechanism of tumor resistance but
a response of the host. This mechanism, mediated by the biomarker–host interaction and not by the tumor, may signicantly interfere with the response of the tumor itself to the
biologically targeted drug.
Considering the growing number of biological drugs targeting known molecular mechanisms that research makes
available to clinical oncology, the issue of measuring biological targets also in blood is becoming crucial. In fact,
despite initial responses, the onset of resistance occurs at
variable time intervals for most of these novel anticancer
agents. The determination of target biomarkers in the primary tumor tissue has the limitations described above, the
most critical of which is the possible occurrence of tumor
phenotype variations, either incidental or induced by the
therapy as a mechanism related to resistance. Frequently,
accessing a metastatic lesion to conrm the actual biological
characteristics of the malignancy is complex, invasive, and
sometimes ethically unsustainable. In this scenario, the possibility of measuring the biomarkers of interest in the blood
is a priority for research and clinical practice. Numerous
studies address this issue by determining the target biomarkers in circulating tumor cells (CTCs) or by examining biomarkers associated with circulating nucleic acids.
Collectively, this approach is referred to as “liquid biopsy,” a
topic covered in another chapter of this textbook. To be
noted, the study of circulating protein biomarkers should not
be seen as an alternative to the study of nucleic acid-derived
components but as a complementary approach. Biological
drugs do not act on nucleic acids but on protein targets.
Moreover, the onset of resistance is not necessarily sustained
by a variation in the mutation of interest but may be due to
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M. Gion et al.
the activation of alternative signaling mechanisms, made
available either by the biological redundancy of tumor cells
or by molecules with a signaling function (cytokines,
angiogenesis factors, etc.) expressed in the tissue
microenvironment.
The microenvironment has attracted attention in recent
studies attempting to identify predictive biomarkers for a
new class of drugs targeting immune checkpoints such as
cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed cell death 1 receptor (PD-1), and programmed cell
death ligand 1 (PD-L1), which has revolutionized the treatment of several solid tumors. These drugs act through an
indirect stimulation of the immune system against neoplastic
cells by inducing consistent and, generally, durable antitumor responses. These treatments exert profound effects on
immune cells within the tumor microenvironment mainly by
recruiting tumor-inltrating leukocytes and modifying the
production of inammatory and immune-related factors,
which may spread into the circulation, becoming possible
disease markers. Given the cost of these drugs and the need
to personalize therapies, more progresses with predictive
markers are crucial. Therefore, the key challenge of immune
checkpoint inhibitor therapies is the identication of reliable
markers to guide disease management decisions and to
advance in the understanding of the mechanisms of resistance to immunotherapy. Numerous biomarkers have already
been tested for their ability to predict response to immunotherapeutic treatments. Although the cancer immunotherapy
eld is still evolving, tissue biomarkers such as PD-L1, mismatch repair, and microsatellite instability testing have
already helped guide the use of US Food and Drug
Administration-approved therapies.
A systematic analysis of the literature shows that several
circulating inammatory or immune-related proteins have
also been implicated in the checkpoint immunomodulation
and in the mechanisms underlying tumor pathogenesis or
progression, including inammatory cytokines, growth factors, C-reactive protein (CRP), and soluble PD-L1. Other
circulating biomarkers that have been investigated in this
context are circulating tumor DNA (ctDNA), circulating
tumor cells (CTCs), circulating microRNAs and exosomes,
and peripheral blood cells. However, a clear association
between circulating biomarkers and clinical responses, resistance, and toxicities to immunotherapy remains to be shown.
As mentioned above, regarding the integration between
the analysis of biomarkers in tissues and blood, the need for
integrating the study of the different types of circulating biomarkers must be considered when designing research strategies. From the long history of biomarkers, we have learned
that considering in a competitive way body districts, biological matrices, molecular classes, or technological approaches,
signicantly slows the translation of innovation to clinical
practice. In the face of the impressive development of knowl-
edge and technologies, reduction of mortality of advanced
solid tumors is still unsatisfactory. To enhance the clinical
usefulness of circulating biomarkers, it is crucial to move
toward a comprehensive vision, to encourage and reinforce
complementarity of knowledge and technologies, and to
share research priorities.
Box 33.1 Circulating biomarkers: general directions
– Biomarkers recommended for diagnostic purposes
should not be used as a standalone test but always in
association with imaging techniques and followed
by biopsy in case of conrmed suspicion.
– Biomarkers cannot be used as a standalone test for
therapeutic decisions in any clinical scenario, but
information provided by biomarkers must be conrmed by clinical criteria or imaging techniques.
– A single increased biomarker value in the absence
of symptoms or instrumental evidence of disease
should be conrmed by a subsequent determination
before any further clinical decision.
– When monitoring a patient, serial biomarker deter-
minations should be performed with the same
method and preferably by the same laboratory.
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Liquid Biopsy
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MarcelloCiaccio
34
Introduction
Two types of tumor-derived biological material can be found
in the blood: circulating tumor cells (CTCs) and circulating
tumor DNA (ctDNA). This chapter describes the technologies used for their isolation and molecular characterization,
their potentiality in clinical-diagnostic terms and the possible impact on patient outcomes. Developing technologies for
isolationg and analysing CTCs and ctDNA has allowed us to
deepen our knowledge of tumor biology and outline possible
applications in the diagnostic eld. Early cancer diagnosis
and early identication of progression represent two very
ambitious goals for liquid biopsy, even if the currently available technologies do not have the required sensitivity for
such applications. To date, the major eld of application for
liquid biopsy is molecular analysis of CTC or ctDNA in the
context of target therapy, especially when traditional biopsy
is not feasible or informative.
Liquid Biopsy
Liquid biopsy refers to the possibility of obtaining tumorderived material, such as cells, proteins, DNA, and RNA,
from a body uid, usually blood. In particular, two types of
biological tumor material can be identied in blood: CTCs
and ctDNA.CTCs are tumor cells that detach from tumor
tissue and circulate through blood vessels. They may arise
from the primary tumor and metastases; in some cases, they
may represent metastatic precursors. CTCs are generally
M. Ciaccio (*)
Department of Biomedicine, Neurosciences and Advanced
Diagnostics, Institute of Clinical Biochemistry, Clinical Molecular
Medicine and Clinical Laboratory Medicine, and Department of
Laboratory Medicine, University Hospital “P.Giaccone”,
Palermo, Italy
Department of Laboratory Medicine, University Hospital
“P.Giaccone”, Palermo, Italy
e-mail: marcello.ciaccio@unipa.it
present in extremely low numbers in the blood of cancer
patients. In addition, the isolation of these cells is complex
and expensive. Therefore, the analysis of CTCs is not performed in clinical practice. Conversely, searching for circulating tumor DNA is widely used in the clinical eld.
However, it is essential to note that liquid biopsy does not
require a cancer diagnosis, for which tissue biopsy is
required. Instead, liquid biopsy can provide crucial information for prognostic or predictive purposes. CtDNA is released
into the bloodstream from tumor cells and lysed CTCs. Once
isolated from patients’ blood, it can be used for molecular
analysis. In particular, ctDNA analysis helps identify the
possible presence of mutations in specic genes that may
make cells sensitive to treatment with molecularly targeted
drugs.
The diffusion of state-of-the-art sequencing technologies
(Next-Generation Sequencing, NGS) and advanced computational methods have recently allowed a more detailed
understanding of the tumor mutational prole by molecular
analysis of CTCs and ctDNA.
There is no doubt that the technological advances will
allow different clinical applications for CTCs and ctDNA,
given their different biological signicance in processes
ranging from tumorigenesis to the most advanced stages of
the disease.
Early studies on CTCs highlighted their negative prognostic signicance in patients with metastatic breast, colon, and
prostate cancer. Although of great therapeutic interest, the
evidence deriving from these preliminary studies was indirect
and did not sufciently support the hypothesis that a more
aggressive therapeutic regimen was more effective in the case
of high levels of CTCs. More recent studies have evaluated
the possibility that specic genetic mutations identied in
CTCs or ctDNA could provide predictive indications on the
efcacy of target therapy. For example, in the case of nonsmall-cell lung cancer, melanoma, and breast cancer, molecular analysis of EGFR, EML4-ALK, BRAF, and PIK3CA
genes in patients with metastatic carcinoma is essential
fortargeted therapy and represents the most established diag-
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Ciaccio (ed.), Clinical and Laboratory Medicine Textbook, https://doi.org/10.1007/978-3-031-24958-7_34
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