Cancer has for years been diagnosed via a biopsy of the
focal lesion tissues. Prostate cancer was diagnosed based upon biopsies of the
prostate, often from samples taken in the "dark", namely needle
biopsies guided by ultrasound, but little else. Melanoma was diagnosed by a
skin biopsy, examining a lesion seen visually and then examined histologically.
Some lesions are examined via immunological tests or other such tests. In
general, this is done on an inspection of specific tissues.
It is also known that primary as well as metastatic lesions
slough off cells, proteins, RNA or DNA, or exosomes of various types which end
up in the blood stream. Primary lesions also use the blood stream as a means to
metastasize, as well as the lymphatic system. Thus there has been an interest
in using what is in the blood stream to see if there is a cancerous growth, to
be used as a prognostic tool, and even to be used as a means to develop some
form of precision therapeutic.
The advantage of sampling the blood is that it is readily
accessible. The disadvantages are multiple:
1. We really have no idea where the cells or components have
come from. Are they primary or a met, are they from a truly virulent cancer or
just an incidental finding?
2. We always face the problem of a cancer stem cell or cell
of origin. Thus what we analyze may be a progeny which is not the driving
factor in the development of the cancer.
3. The "tool" problem is always there. Namely, do
we have tool adequate to ascertain a single cell or cell component to achieve
the desired specificity and sensitivity?
Many of these hurdles are being overcome and the examination
of a individual based on sampling of the blood is now within reach. This is
essentially the field of "liquid biopsy".
Some of the earliest work on cancer cell shedding was done
in the early 1970s and reported by Butler and Gullino who noted:
The rate of tumor cell shedding into efferent tumor blood
was measured in growing and regressing MTW9 rat mammary carcinomas. The
hormone-dependent tumor, grown as an isolated preparation, permitted collection
of all of the efferent blood. Regression was induced by reduction of
mammotropin level in the host. Tumor cells were differentiated from normal
leukocytes by indirect immunofluorescence. Growing tumors shed 3.2 x 106
and regressing tumors shed 4. 1 x 106 cells per 24 hr per g tissue.
Cell shedding rates of
growing versus regressing tumors were not significantly different over a
tumor size range of 2 to 4 g. The number of tumor cells in the arterial blood
was 12-fold smaller than in the efferent tumor blood. It is concluded that: (a)
cell shedding via blood probably plays only a minor role in the total cell loss
by growing MTW9 carcinomas; (b) hormone-induced tumor regression does not
depend on increased cell shedding; (c) tumor cells are rapidly cleared from
circulating blood; and (d) a 2-g MTW9 carcinoma pours enough cells into the
host circulation to transplant the tumor every 24 hr.
Thus there is a continual shedding of tumor cells and the
above result has been verified many times over the past decades. But again,
this shedding is from anywhere the tumor may have resided, and in its journey
in the blood stream we would find it difficult to determine that. However we
have argued elsewhere that it may very well be possible to determine the
location of the cell by its surface markers which often are descriptive of from
whence it came[2].
As noted in the NCI site[3],
the current working definition of "liquid biopsy" is as follows:
A test done on a sample of blood to look for cancer cells
from a tumor that are circulating in the blood or for pieces of DNA from tumor
cells that are in the blood. A liquid biopsy may be used to help find cancer at
an early stage. It may also be used to help plan treatment or to find out how
well treatment is working or if cancer has come back. Being able to take
multiple samples of blood over time may also help doctors understand what kind
of molecular changes are taking place in a tumor.
In a recent paper by Harris et al (2018) the authors note:
Cancer stem-like cells (CSCs) are associated with cancer
progression, metastasis, and recurrence. CSCs may also represent a subset of
tumor-initiating cells, tumor progenitor cells, disseminated tumor cells, or
circulating tumor cells (CTCs); however, which of these aggressive cell
populations are also CSCs remains to be determined. In a prior study, CTCs in
advanced prostate cancer patients were found to express CD117/c-kit in a liquid
biopsy.
Whether CD117 expression played an active or passive role
in the aggressiveness and migration of these CTCs remained an open question. In
this study, we use LNCaP-C4-2 human prostate cancer cells, which contain a
CD117+ subpopulation, to determine how CD117 expression and activation by its
ligand stem cell factor (SCF, kit ligand, steel factor) alter prostate cancer
aggressiveness. CD117+ cells displayed increased proliferation and migration.
Further, the CD117+ cells represented a CSC population
based on stemness marker gene expression and serial tumor initiation studies.
SCF activation of CD117 stimulated increased proliferation and invasiveness,
while CD117 inhibition by tyrosine kinase inhibitors (TKIs) decreased
progression in a context-dependent manner. We demonstrate that CD117 expression
and activation drives prostate cancer aggressiveness through the CSC phenotype
and TKI resistance.
This article highlights several interesting areas and their
convergence.
First, the reassessment of the cancer stem cell especially
as applied to PCa.
Second, the use of CTCs to assess the progress of a disease
and thus establish a reliable prognostic marker.
Third, the identification of CD117 as a specific marker for
an aggressive form of PCa.
The authors provide an interesting platform for this
convergence but it also allows for a window on all three of these areas. CTCs
are receiving more attention. We know that cancer cells leave local sites,
travel through the blood to distant sites. Likewise these distant sites also
slough off cells or parts of cells. We have previously examined this for
oncosomes in prostate cancer a while back[4].
Thus we may ask; why a liquid biopsy at all? Zhe et al give
a fundamental answer:
Despite major advances in research and therapy, cancer
continues to be the second cause of death in the United States, with 1 in 4
deaths due to cancer. Primary tumors rarely have deadly consequences, while
metastatic disease accounts for around 90% of the mortality due to solid
tumors. Therefore, the development of new sensitive methods that allow the
detection of cancer dissemination, most notably in the common carcinomas,
before full blown clinically detectable gross metastatic deposits are
established is of tremendous utility to help physicians in treatment decisions.
Basically a liquid biopsy presages metastatic growth.
Localized tumors, often termed "carcinoma in situ" are just that,
local. The cells may have begun to proliferate but they do so locally and have
not begun to wander into the blood or lymph system.
Liquid biopsies come in many different forms. We outline
them in the figure below and then provide a brief discussion (see Abraham et
al).
1. CTCs Circulating Tumor Cells: CTCs represent intact,
viable non-hematological cells with malignant features that can be isolated
from blood
2. ctDNA Circulating tumor DNA: Circulating cell-free DNA
(cfDNA) are small DNA fragments found circulating in plasma or serum, as well
as other bodily fluids.
3. cfRNA Circulating Free RNA: Tumor cells actively release
several species of cfRNAs, into the blood including non-coding RNAs
4. Exosomes: High levels of exosomes are found in several
bodily fluids from cancer patients
DomÃnguez-Vigil et al present
a recent update as to what can be the basis for detection of cancers via
hematological sampling. They note:
Circulating tumor DNA (ctDNA): DNA is continuously
released in fragments into the circulation through processes such as apoptosis
and necrosis by both normal and cancerous cells. When released irrespective of
cell of origin, it is typically referred to as cfDNA (cell-free DNA); but when
released specifically by cancer cells, it is mostly referred to as ctDNA
(circulating tumor DNA). Among the molecular characteristics of ctDNA are that
it may harbor mutations, CNVs, methylation changes, or integrated viral
sequences associated with the tumor
Circulating tumor cells (CTCs): CTCs have been discovered
for Asworth in 1869 during an autopsy of a patient who had metastatic cancer.
They are cancer cells that detach from a primary or metastatic tumor site and
are present in the circulation. Clinical evidence indicates that patients with
metastases have 1–10 CTCs per mL of blood and they are rarely found in
clinically healthy people or in people with nonmalignant tumors. CTCs have been
detected in different types of cancers, such as breast, ovarian, prostate,
lung, colorectal, hepatocellular, pancreatic, head and neck, bladder, and
melanoma
Exosomes: Exosomes are small round vesicles, 30–120 nm in
diameter, and of endosomal origin carrying RNA, miRNAs, DNA, and proteins that
are released by multiple cell types (including tumor cells) into the
extracellular environment. Exosomes may mediate some form of communication
between cells, being internalized by other cells
miRNAs: MicroRNAs or miRNAs are small molecules of
non-coding RNA, between 19 and 24 nucleotides in length, that act as regulatory
molecules of gene expression, exerting function by hybridizing to inhibit the
translation of mRNAs of its target genes. Differential expression of miRNAs in
patients with cancer has been described.
The area of "liquid biopsies" is an active and
exciting area of research. However it still presents a multiplicity of
challenges. We briefly discuss some these here.
This is an interesting and strongly visual result. However,
there are several observations:
Mutations: Cancer cells are continually mutating. Mutations
results oftentimes in new surface receptors due to the changes in the internal
proteins. The cell surface receptors may respond differently to the passage
through such a cellular membrane. Thus this model may be reflective of itself
but not of reality.
Localization: One of the most intriguing things about
cancers is the localization of the metastases. Why, for example, do we so often
see prostate cancer go to the bone, melanoma to the brain, across the blood
brain barrier, and the same with so many other cancers. There is a
predisposition to transfer at specific sites. How does this approach deal with
such localization effects?
Stem Cells: Stem cells are a potential significant factor in
understanding metastasis. One question is; do stem cells move as easily as
others or more so? Or, are stem cells just active wherever they are and their
products are carried through the blood stream to sites where they can continue
cellular proliferation, and possibly induce a new set of stem cells there?
In and Out Flows: One of the questions one must ask when
looking at cancer cells in the blood, as has been done recently, is if the cell
is coming or going? Namely is the cell going from a source site, a primary, to
a remote or metastatic site, or from an already metastatic site to another new
one? The tagging of such cells would be important. The understanding of the
genetic changes would also be of critical importance.
Biochemical Drivers: The nature of cell surface markers,
receptors and the like, often dominate how the cells behaves, interacts with
the ECM, and can move to the blood system and exit from it as well. We have
argued that the cancer cell just flows and diffuses in the blood system and
that there is no growth. That is just gross speculation but it is open to
debate. Moreover the interaction of the cell with the cell way, and the
localization effects of the cell way by organ specificity may be an attractive
basis for organ specific metastasis. Or possibly not. But, having all these
elements at play in vivo is better than in vitro.
Immune System: Then also is the impact of the immune system
as the cells flow through the vessels. The cells are in a massive amount of
immune system interactions, and how does this impact the cells?
These are but a few of the unanswered questions elicited by
this paper. The simulation is well worth looking at the paper, but taking its
results as fact is stretching it a bit too far.
It would appear that a great deal of progress has been made
yet there are still many significant challenges. As Kaiser has recently noted:
A team of researchers has taken a major step toward one
of the hottest goals in cancer research: a blood test that can detect tumors
early. Their new test, which examines cancer-related DNA and proteins in the blood,
yielded a positive result about 70% of the time across eight common cancer
types in more than 1000 patients whose tumors had not yet spread—among the best
performances yet for a universal cancer blood test. It also narrowed down the
form of cancer, which previously published pan-cancer blood tests have not. The
work, reported online today in Science, could one day lead to a tool for
routinely screening people and catching tumors before they cause symptoms, when
chances are best for a cure. Other groups, among them startups with more than
$1 billion in funding, are already pursuing that prospect.
The new result could put the team, led by Nickolas
Papadopoulos, Bert Vogelstein, and others at Johns Hopkins University in
Baltimore, Maryland, among the front-runners. “The clever part is to couple DNA
with proteins,” says cancer researcher Alberto Bardelli of the University of
Turin in Italy, who was not involved in the work. The researchers have already
begun a large study to see whether the test can pick up tumors in seemingly
cancer-free women.
Yet there are reservations. Kaiser concludes:
For those who test positive twice, the next step will be
imaging to find the tumor. But that will bring up questions raised by other
screening tests.
Will the test pick up small tumors that would never grow
large enough to cause problems yet will be treated anyway, at unnecessary cost,
risk, and anxiety to the patient?
Papadopoulos thinks the problem is manageable because an
expert team will assess each case. “The issue is not overdiagnosis, but
overtreatment,” he says. Still, others working on liquid biopsies say that it
will take time to figure out whether widespread screening of healthy people
with a universal blood test can reduce cancer deaths without doing harm. “If
people expect to suddenly catch all cancers, they’ll be disappointed,” says
cancer researcher Nitzan Rosenfeld of the University of Cambridge in the United
Kingdom. “This is exciting progress,” he says. “But evaluating it in the real
world will be a long process.”
Namely Kaiser does reflect the reality of assuming that
liquid biopsies are a pending reality. As we have noted, there may be a chance
for prognostic use in already metastatic disease. Will liquid biopsies identify
indolent cancers, will its use result in putative diagnoses that result in
costly tests and procedures but to no avail.
Overall as a physician, one would like to know what lesion
is where and how large and how aggressive. Then using the therapeutic tool box
one could possibly treat the lesion.
As noted above there is considerable interest in
"liquid biopsy" approaches but yet there are reservations as
well. As Fouad and Aanei have noted:
Once in circulation, circulating tumor cells (CTCs) are
exposed to harsh selective conditions and must devise adaptive techniques.
Examples include platelet coats shielding from shear forces and
immune-clearance, and metabolic rewiring blunting oxidant stress. Serving as a
“liquid biopsy”, isolated CTCs could provide means for cancer screening,
estimation of metastatic relapse risk, identification of targetable components,
exploring tumor heterogeneity, and monitoring therapeutic response. Multiple
challenges still stand in the way and will need to be addressed before clinical
utilization.
CTCs can be a powerful marker. Yet they leave many questions
unanswered.
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