Changing Nature of Diagnosis and Treatment of Cancer
Abstract
Traditionally, cancer has been diagnosed / classified by pathologist
under the microscope. Different types of cancers have been identified in
relation to the location where they are first observed. Also, treatment
and prognosis of cancer has been in relation to its histopathology and
pathology analysis. Misdiagnosis and mistreatment have resulted from
this approach which has increased the suffering and death of patients.
Newer diagnosis and treatment methods are based on the molecular
analysis of the tumor genes and identification of mutations which are
directly related to the tumors. Under the new system, tumors are
classified and treated according to their mutation type and not based on
their location. Among the treatment options, immunotherapy relies on
such approach.
Introduction
The immune system oversees body's protection against internal and
external maladies. As we age, the immune system undergoes changes that
are brought about through signals that are produced within the body
itself. These changes affect the immune system's ability to protect the
body against the diseases. On occasions, even the signals that are sent t
through alterations in the genes to the cells in various body organs,
weaken or get misinterpreted, directing the cells to become diseased
including cancerous. The alterations in the genes include addition or
deletion of DNA pieces or changes in the number of DNA copies. The
tumors that result from such alterations may contain certain segment of
DNA, or multiple copies of the whole gene. Alternatively, a single base
in the DNA may be chemically modified via methylation or other
reactions. At the end, in a single tumor, an average of 50 different
mutations may be created through various processes which compared to the
total number of genes in the body is very few. Our body has 3 billion
bases in its genes of which only a few may be modified, replaced, or
deleted due to such mutations. Throughout our life, many spontaneous
mutations also occur in our genes. All and all, only a few of the all
the mutations may lead to serious conditions like cancer [1].
When created, each tumor type carries specific mutations, some with
higher number of mutations and some with lower number [2]. In a series
of high-throughput sequencing (HTS) studies, four genes were found to be
altered in over 20% of the tumors [3,4]. Of course, all mutations do
not lead to cancer. Cancer results only when mutated cells can
circumvent the normal defensive mechanisms of the body. These defensive
mechanisms catch and correct most replication mistakes, leaving only
some mutations to lead to cancer. Some common defensive mechanisms that
prevent formation of cancer include: TP53 tumor suppressor gene, DNA
damage gene, and checkpoint genes, but other genes including
anti-oncogenes, and caretaker genes may also participate. Most often,
mutations are corrected by specific enzymes controlled by tumor
suppressor genes [5] but occasionally, they are not corrected, and the
alterations will carry on for many generations until they take over the
whole cellular machinery. In the process of becoming cancerous, the
cells first go through the pre-cancerous stage and then to the cancerous
stage characterized with uncontrolled growth. Once they have passed
through the pre-cancerous stage, cancer cells display enormous genomic
instability [5], changing their genome more and more as they grow. The
mechanism of passage from precancerous to cancerous stage may involve
changes in the caretaker genes (mostly observed in hereditary cancers)
and production of new proteins through translation and transcription
that are responsible for changing the normal cellular pathways.
The initial mutations may also be hereditary, or may be caused by
environment, lifestyle, genetic, food, or other factors called
pathogenic variants. Thanks to advancements in genetic techniques such
as CRISPR, today we have gained the ability to detect and avoid genetic
diseases by artificially editing the mutated parts of the genes even in
the embryo, during pregnancy, or right after birth. But this and other
techniques despite their great potential are still in their infancy and
are being rapidly developed in many parts of the world. It is important
to note that knowing how it develops, we can see why cancer is not just
one disease but a family of hundreds of diseases with huge genetic
variability. The variability of cancer necessitates the generation of
huge amount of data and this by itself adds to the difficulty of
managing the cancer. Finding one damaged DNA among the 3 billion DNA
bases in the human genome is time consuming and costly. Today, this is
done in a matter of few days at a reasonable cost. Of course, when a
mutated gene is discovered in a tumor, effective treatment may or may
not be available because effectiveness of the treatment is case specific
and if a treatment works in one case, it may not work, or it may have
to be modified to make it work in another case. Variations exists among
the people who even have the same cancer.
The New Paradigm
So far, cancer has been diagnosed and attacked as a disease of the
specific organ where it is first detected, e.g., cancer of the lung,
cancer of the liver, cancer on the head and neck, etc. In the new
paradigm, cancer is look at not as an organ specific disease but as a
genetic malfunction that may appear at any part of the body. Under this
new system, cancer cells are not classified or categorized by a
pathologist under the microscope but studied and diagnosed by a
geneticist and/or pathologist trained in the genetics. Treatment too
will be based on the type of genetic mutations in the cancer. This
approach to diagnosis and treatment for cancer will eventually be
applied to many diseases that result from genetic malfunctions and
mutations. Accordingly, it is expected that the diagnosis and treatment
of cancerous and many non-cancerous [6] genetic maladies will be
organized and performed based on the specific genetic profiles of those
diseases which are obtained through massive parallel sequencing (MPS) of
their genomes. Both RNA and DNA will be assayed and targeted for
treatment [7].
In the case ofcancer, recent studies have established a correlation
between the sequence of the tumor genes, the prognosis for the cancer,
and survival of the patients [8]. Among the 3281 tumors that were
studied, according to TCGA, 93% had at least one mutation [9]. The
relation between the molecular profile of tumors and the clinical
manifestation of the tumors have been described for several tumors
including localized prostate cancer [10]. Also, a relationship between
the metastatic behavior [11,12] of cancers and the tumor genetic profile
has been established [13]. In the diagnosis of cancer based on its
tumor gene profile, one looks at the genetic makeup of the tumor and
classifies the tumor accordingly. In this approach, "driver mutations",
mutations that are responsible for the cancer development are
identified. Then, tumors are targeted with specific drugs that have been
designed to attack those specific mutations. Using this approach, for
both diagnosis and treatment of cancer, we look at the molecular makeup
of the basic cell constituents, DNA, thus gradually moving away from the
current practice of diagnosis and treatment based on morphology and
histopathology.
Of course, when cancer cells become resistant to treatments, to
overcome the resistance, we again must resort to molecular changes that
are responsible for the resistance and design treatments which only
target those molecular changes. In resistance, initial "driver genes"
may become dormant and new "driver genes "take over their role.
Sometimes, it is not the genes, but the way genes are expressed that
leads to the appearance of cancer or resistance to treatment [14].
Biology at the molecular level is chemistry but diversity among the
molecules and their roles in the biological systems makes understanding
the relationship between the two challenging. These challenges stem from
the high number in the variety of different diseases associated with
cancer, and the heterogeneity of the mutations amongst the various
tumors and within each tumor type as well as the enormous amount of data
that must be generated and the cost involved. Unfortunately, a higher
level of mutagenic heterogeneity is associated with a worse prognosis
[15]. Advantages of diagnosis and treatment based on genetic profiling
include avoiding misdiagnosis, requirement for smaller samples, and the
possibility of using skin or blood samples for diagnosis (Figure 1).
Figure 1.
Correlation Between Pathology and HTP Gene Profiling
One major benefit of targeting (mutationally similar) tumors based on
the genetic makeup of their mutations rather than their location is
that during the testing of the drugs, clinical trials don't need to be
repeated for each organ that share the same type of mutation. Using the
current classifications based on histology and histopathology, many
cancers including breast cancer have been misidentified and misdiagnosed
[16]. This has led to patients suffering or dying unnecessarily. It is
hoped that using the newer method of gene profiling, and designing
treatments based on the mutation profile of the tumors, misdiagnosis and
inappropriate treatment will be reduced. In several studies, good
correlations have been shown between the traditional methods of cancer
diagnosis and prognosis, HTP [17,18], and targetable mutations [6].
Comparison of the cost and laboratory techniques between the two
approach as well as sample handling have also been made [2]. To
accurately correlate the traditional methods of diagnosis and treatment
based on pathology and the new gene profiling, profiles of more genes
must be defined using methods that may require synthesis of DNA pieces
or strands. Today, in the lab, we can synthesize the whole genomes of
small viruses and bacteria but synthesizing the whole human genome is
way more complicated and challenging. Until the time that we can
synthesize the genome of humans, we can provide help for patients by
editing their mutated genomes [19-21]. For editing, the genetic
"mistake" inherited or created because of mutation is detected and
corrected as if it were a word document [22].
Immunotherapy
Immunotherapy involves using T-cells, normally in charge of the
body's defense, to attack and destroy foreign bodies including cancer
cells. In cases where T-cells attack non-cancerous "self" cells, the
body will normally get rid of those T-cells, or else, they will cause
autoimmune disease. To avoid being detected as a foreign body, the
cancer cells use a variety of tricks to show themselves as "self" rather
than foreign to the T-cells and thus avoid being detected and
destroyed. In immunotherapy, either the T-cells or the cancer cells are
modified at the molecular level so that the cancer cells can be
selectively identified and destroyed by the immune cells. The
modifications are necessary because without them, T-cells are not able
to recognize cancer cells as foreign cells to attack them. Two methods
of modifications are currently prevalent
- a) Using checkpoint inhibitors to remove the proteins or peptides
that cancer cells produce to fool the T-cells into thinking that they
are not foreign but "self" cells. Keytruda,a checkpoint inhibitor was
tested in 49 patients who had not responded to other drugs, 40% with
colon cancer and 48% with other types of cancers showed dramatic
shrinkage or complete disappearance of the tumors. Keytruda and Yervoy
are antibodies approved by the FDA that increase T-cell activity towards
tumors by blocking proteins that prevent T-cells from being active
against the tumor. Antibodies work by selectively attaching themselves
to specific target proteins and taking those specific proteins out of
the circulation and remove them from accessibility. It is estimated that
20% of the cancers respond to checkpoint inhibitors. However, of those
that respond, some may develop resistance due to mutation or other
mechanisms and make themselves invisible to the T-cells.
b) Modifying the immune system T-cells that are not able to recognize cancer cells as foreign cells so that they are able to recognize the cancer cells as foreign and attack and destroy them. An example of that are the chimeric antigen receptor (CAR) T cells or modified CAR-T cells which can recognize and attack the cancer cells as foreign cells. In practice, through genetic engineering, T-cells are modified so that they carry a protein (called CD19) that binds specifically to the cancer cells and capture them through antigen-antibody interaction. The genetically modified T-cells are grown outside the body before they are given to the patient for treatment. This technique which has worked for leukemia can evoke severe body reaction because it involves elevation of cytokines related to amplified immune response. In the (CAR) T cells method tested recently, T-cells were removed from a leukemia patient, were genetically modified to recognize cancer cells as foreign, and then they were given back to the patient and allowed to fight the cancer [23]. Here, the T cells were engineered to destroy any cell that has the protein CD19 on their surface, in the body of the leukemias and lymphomas patient. The altered T cells are called CAR-T cells.
In one form of CAR-T-cell immunotherapy called TIL, T-cells that are
naturally able to recognize and attack cancer cells but are limited in
number, are collected from the surfaces of the solid tumor tissues. They
are then grown ex-vivo, outside the body of the patient in huge numbers
and then given back to the patient so that they attack and kill the
cancerous cells. This method has been successful at least in one case of
cholangiocarcinoma [24] at NIH. Cancer vaccines may also be used to
trigger the action of T-cells that remain inactive against the cancer
cells. Antibodies may be used to detect and capture either the cancer
cells or the immune cells. Under ideal circumstances, tumor cells will
have peptide or protein residues hanging off them that can act as
antigens which can be recognized by T-cells as foreign bodies and
attacked through the antigenic response system. Tumors develop many
mutations. Some of those mutations lead to production of entities
(antigens) that extend outside their cells, in the form of a peptide.
For example, the EGFRvIII mutation is one such mutation
that extends a peptide piece outside the cell which provides an
opportunity for raising antibodies against them. Such antibodies can
then be used as drugs to help kill the tumor cells. Once T-cells
recognize the cancer cells with antigens, they proceed to capture, lyse
and kill those cancer cells and thus destroy the tumor. Of course, there
are several types of T-cells each of which have different defensive
duties which they perform by utilizing different mechanisms.
One limitation of the diagnosis and treatment of cancer based on
genetic profile is that the types of mutations abound and not all
mutations lead to cancer. In addition, once formed, cancer cells undergo
further mutations. As a result, even diagnosis/ treatment based on
tumor mutation is not so simple. So far, early immunotherapies in the
Clinique Based on Cancer Genome Atlas (TCGA) [25], have shown that best
outcomes may become reality if treatments are designed for each
individual case (personalized medicine). Many centers have initiated
such personalized trials but developing drugs for individual patients by
pharmaceutical companies is not profitable and practical although the
FDA has indicated that it will approve such drugs. An additional
complication is that often multiple changes to the gene are necessary
for the cancer to develop. Alternatively, the changes may take 10, 20,
30, 40, 50 years, or a lifetime to develop into cancer.
Common Mutations and their Relevance to Inherited Cancers
When a patient inherits a gene mutation, the risk of hereditary
cancer is higher in that patient than average population. Some
hereditary cancers appear at certain designated time at one's life.
Often one or more family members share the hereditary cancers. In one
case, a female patient, developed cholangiocarcinoma at the age of 61,
her father died of pancreatic cancer at the age of 58.But in a family of
8 children, only one of her brothers developed lymphatic cancer at age
50. Overall, the risk for hereditary cancers are way lower than the risk
for non-hereditary cancers. The non- hereditary cancers which result
from gene mutations caused by food, hormone changes, or environmental
factors, make up the bulk of cancers today. Because about 5-10 percent
of the cancers occur through inheritance. Genetic testing can be
performed to identify the inherited mutations. Mutations identified in
the hereditary cancers include BRCA1 and BRCA2, PTEN. These mutations
increase the risk for breast and thyroid cancer, the STK11 mutation
increases the risk for colon, breast, and pancreatic cancer, and the TP
53 mutation increases the risk for breast, sarcomas, and brain tumors.
Lynch syndrome which is hereditary involves mutations in MLH1, MSH2,
MSH6, or EPCAM genes and is associated with colon, uterine, ovarian, and
gastric, CNS, and pancreatic cancers. People who have the hereditary
Lynch syndrome have a 70% risk for colon cancer. They are also at risk
for ovarian, and endometrial cancers. All this is due to a defect in
their MMR genes. These genes are in charge of correcting mistakes that
may occur in DNA replication. Cancers with MMR defects respond to
immunotherapy.
Mutations in the PTEN gene increases the risk for melanoma and
breast, uterine, thyroid, colon, and kidney cancers. Mutation in TP53
(Li-Fraumeni syndrome) gene is associated with increased risk for colon,
bone, pancreatic, liver, brain tumors and leukemia. Mutations in CDH1
gene increases stomach and lobular breast cancer. Gene panel tests are
nowadays available for various types of syndromes that lead to known
tumors. A Breast or ovarian cancer test panel for example will look for
mutation in the BRCA1 and BRCA2 genes and other relevant genes. When
multiple mutations are involved in one's cancer, to stop the cancer,
inhibitors for all those mutations must be administered. For example, in
colon cancer, both BRAF mutation and EGFR mutation may be involved in
which case inhibitors for both mutations must be administered. Normally,
EGFR mutation is found in lung cancer. About 30-45% of patients with
melanoma have BRAF V600E mutations but less than 2% of them may have
non-small lung cancer. Genetic testing in the past 2-5 years has opened
the door for finding even rare mutations and drugs that have shown great
promise and significant tumor shrinkage or complete remission. In
addition to inherited mutations, in recent years, testing on somatic
mutations has dramatically increased. They help identify those therapies
that might work against a tumor. But because all the possible mutations
have yet to be identified, each testing may identify new mutations. In
terms of mechanism, similar pathways may be involved in the sporadic
cancer as in hereditary cancers. For example, in the non- hereditary,
sporadic breast cancer, the levels of BRCA2, a tumor suppressor, which
is involved in the repair of the DNA, goes down as mutation is observed
in the sporadic breast cancer. This in turn leads to proliferation and
invasion [26].
Personalized Cancer Treatment
In personalized therapy, the mutational as well as transcriptional
profiles of a person's tumor is first obtained. Then, therapies are
designed that specifically attack that very profile of the cancer.
Unfortunately, now, enough drugs are not available to target all the
detected mutations. But the method of targeted therapy has been proven
successful in many cases [27]. NCI-MATCH trial program sponsored by NCI
started in 2015. The program has sequenced the tumors from 6000
patients. In this trial, 21 drugs alone or in combination are given to
the patients based on the mutations and sequence in their genes. A
similar trial is run by Novartis which started in 2013. This trial tests
15 types of cancers in 600 patients. When gene of the adenocarcinoma of
the tongue was sequenced, mutation in PTEN gene as well as
overexpression in RET gene was observed. Treatment with the RET
inhibitor, Sunitinib, stabilized the cancer. Also, in a thyroid cancer
case, mutation in mTOR was observed which prompted treatment with mTOR
inhibitor. In other studies, patients were given drugs that matched the
identified mutation. Of the 11 patients, 3 responded and 4 became
stable. Other treatments based on mutation include:
a) Larotrectinib is a drug that targets TRK gene fusion which causes
about 5000 cancers each year including lung and colon cancers. Complete
remission by treatment with this drug was observed in 17 cases.
b) Vemurafenib which was expected to work against cancers with BRAF
mutations such as melanoma, colon, and thyroid, works on melanoma, and
skin cancer but not on colon cancer. However, if EGFR inhibitor (common
to lung cancer) is added to it, it then works against colon cancer also.
Researchers are now testing broadness of the effectiveness of various
inhibitors. For example, they test PARP inhibitors (which have shown
activity against BRCA-muted ovarian cancer), against cancers related to
other mutation.
Synthetic Lethality
In this method, vulnerability of the cancer cells is discovered, and
those vulnerabilities exacerbated by treatments that in effect are
additional attack [28] on the cancer cells. For example, cells use two
repair mechanisms to repair any damage to their DNA, if one of those two
repair mechanisms is hampered by becoming cancerous, and cells depend
only on the second repair pathway for their survival, then blocking that
only pathway will lead to their demise and thus destruction of the
cancer. Mutations in BRCA genes disables one of the two pathways that
the breast cancer cells need to repair their DNA. So, they are left with
only one DNA repair pathway for their survival namely, Poly
(ADP-ribose) polymerase (PARP) which repairs single stranded DNA.
Because the cells are already vulnerable, attacking this second pathway
by using inhibitors of the PARP, will lead to the cell death [29]. Drugs
that inhibit the PARP enzyme include Olaparib, rucaparib, and
niraparib.
CRISPR-Cas9
CRISPR-Cas9 technology is a new tool that allows scientists working
in genetics to cut and paste pieces of DNA. This process which is
biologically conducted by enzyme, is not too different from the cut and
paste used in fixing a word document. The process allows the creation of
new strands of DNA which can code for creation of new proteins with
varied functions and properties. The technology can be used to modify
cancer cells for ease of targeting. It can also be used to modify the
T-cells in charge of body’s defense so that they can recognize and
target the cancers cells. In addition, the technology can be used to add
or delete specific traits to plants and or animals. In plants, for
example, the lost favor of the tomatoes can be revived or tomatoes with
high yields can be generated through CRISPR technology. In animals,
animals possessing specific characteristics can be created. In nature,
the CRISPR-Cas system oversees providing immunity for bacteria against
viruses and plasmids. CRISPR-Cas9 is based on CRISPR-Cas system. The
protein Cas9 is an endonuclease that uses a guide sequence to form base
pairs within target DNA sequence. This allows for introduction of a
specific double stranded break in the DNA [30,31]. The technique allows
for efficient targeting, editing, and modifying of cells and organs
which can influence the modification and regulation of their behaviors.
The technology not only can help with correction of mutations, to
prevent cancer, it can also help prevent many other inherited diseases.
In addition, the technology can be utilized to design drugs, generate
specifically engineered cells, organs, organisms, and animals, and
plants. In research, generation of models that contain multiple
mutations is necessary [32]. To create an animal cancer model with
several genes, currently, breeding is used which time is consuming.
CRISPR/Cas9 on the other hand can introduce several mutations in one
step. For example, in ES cells, it can be used to introduce over five
genes in the cells [33-36]. In practice, CRISPR uses a guide RNA to
direct the Cas9 enzyme (light blue) to target DNA sequence (Figure 2).
Once there, every time, Cas9 finds a protospacer-adjacent motif sequence
(red) in the DNA, it will bind to and cut both strands. This process
primes the gene sequence for editing [37-45]. CRISPR is only 5 years old
but because of extraordinary potential value in helping patients, it is
being tested in the clinic. In 2018, several companies are for the
first time taking the technique to the clinic. However, currently, the
clinical studies focus on fixing mutations that lead to sickle cell
disease. In this disease, red blood cells take certain shapes that
prevents them from getting out of the blood vessels for delivering
oxygen to the tissues.
Figure 2. 
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