1150772
research-article2023

CIX0010.1177/11769351221150772Cancer InformaticsQuraish et al

An Overview: Genetic Tumor Markers for Early
Detection and Current Gene Therapy Strategies
Reeshan ul Quraish, Tetsuyuki Hirahata, Afraz ul Quraish
and Shahan ul Quraish

Cancer Informatics
Volume 22: 1–13
© The Author(s) 2023
Article reuse guidelines:
sagepub.com/journals-permissions
DOI: 10.1177/11769351221150772
https://doi.org/10.1177/11769351221150772

Hirahata Gene Therapy Laboratory, HIC Clinic, Tokyo, Japan.

ABSTRACT: Genomic instability is considered a fundamental factor involved in any neoplastic disease. Consequently, the genetically unstable
cells contribute to intratumoral genetic heterogeneity and phenotypic diversity of cancer. These genetic alterations can be detected by several
diagnostic techniques of molecular biology and the detection of alteration in genomic integrity may serve as reliable genetic molecular markers
for the early detection of cancer or cancer-related abnormal changes in the body cells. These genetic molecular markers can detect cancer
earlier than any other method of cancer diagnosis, once a tumor is diagnosed, then replacement or therapeutic manipulation of these cancerrelated abnormal genetic changes can be possible, which leads toward effective and target-specific cancer treatment and in many cases,
personalized treatment of cancer could be performed without the adverse effects of chemotherapy and radiotherapy. In this review, we describe
how these genetic molecular markers can be detected and the possible ways for the application of this gene diagnosis for gene therapy that can
attack cancerous cells, directly or indirectly, which lead to overall improved management and quality of life for a cancer patient.
Keywords: Gene therapy, immune cell therapy, DNA vaccine therapy, cytokine therapy, tumor suppressor gene therapy
RECEIVED: July 5, 2022. ACCEPTED: December 24, 2022.
Type: Review

Declaration of Conflicting Interests: The author(s) declared no potential
conflicts of interest with respect to the research, authorship, and/or publication of thisarticle.

Funding: The author(s) received no financial support for the research, authorship, and/or
publication of this article.

CORRESPONDING AUTHOR: Tetsuyuki Hirahata, Hirahata Gene Therapy Laboratory,
HIC Clinic #1105, Itocia Office Tower 11F, 2-7-1, Yurakucho, Chiyoda-ku, Tokyo 100-0006,
Japan. Email: hirahata@hicc.jp

Introduction

into the body of a patient to attack and kill the cancer cells.7-9
In this review, we explain briefly about gene tests such as concentration and fragment length of free DNA, DNA mutation
detection test, DNA methylation test, and Gene expression
test for early detection, diagnosis, and prognosis of cancer, we
also describe different types of gene therapies performed toeither directly attack cancer cells or to induce the immune system of a cancer patient to fight cancer. In the field of oncology,
molecular genetics of cancer is becoming an important tool for
screening, surveillance, treatment, and management.
Future research and improvements in cancer genetics will
increase the ability to precisely and cost-effectively diagnose
and treat cancer patients. The purpose of this report is topresent the clinical work performed in our laboratory (HIC
Clinic) and to review it with the current research in this area.

In our previous article, we described how liquid biopsy can beused for the detection of genetic tumor markers for the early
diagnosis and prognosis of cancer.1 In this current article, wewill review various nucleic acid based biomarkers for genediagnosis and clinical methods for gene therapy to cure cancer
disease. Cancer is a multi-factorial genetic disorder and a complex group of diseases in which cells grow out of control and do
not die after being damaged or useless, and show immortality.2
There are many factors involved in the process of cancer generation such as; environmental factors, lifestyles, and infections,which can then lead to abnormalities in cancer-related genes at
a molecular biology level. Abnormal changes in body cell molecules (genes) and their products can be detected and serve as
genetic tumor biomarkers.3,4 These direct tumor biomarkers ofgenetic origin offer an earlier and more precise diagnosis ofcancer than the conventional protein-based tumor markers.4
Another advantage of these gene-based tumor biomarkers isthat they can also be used to see the prognosis of the disease if
one or more of these biomarkers are monitored for changes
during and after the therapy.5 In addition, molecular abnormalities in the genes or DNA offer targets (cancerous cells)
that can also be utilized to develop a specific gene-drug which
recognizes and attack only such abnormal targets (cancerous
cells) to cure cancer without producing side effects at all or
minimum.6,7 These genetic biomarkers can also be used toenhance the immune cell therapy against cancer, therefore,
more effective and specific immune cells can be engineered in
the laboratory by the application of gene transfer technology ,consequently, these immune cells become effective cancerattacking cells, then these engineered cells can be re-introduced

Gene Test for the Early Detection of Cancer

As mentioned above, there are cancer-producing alterations ingenes and these abnormalities can be detected as genetic
tumor markers to diagnose the presence of cancer in the
body.3,10 The progression of cancer involves multiple genetic
and epigenetic events that disrupt the balance between cell
division and apoptosis. Genes that affect cancer progression
are known as cancer driver genes,11 which can be classified astumor suppressor genes (TSGs) and oncogenes (OGs) basedon their roles in cancer progression.12 Oncogenes are usuallyactivated by gain-of-function mutations that stimulate cell
growth and division. Whereas, tumor suppressor genes arerecessive, anti-proliferative, and frequently found inactivated or
mutated in cancer.13 These genes are inactivated by loss-offunction (LoF) mutations (insertions/deletions and nonsense

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Cancer Informatics ﻿

2Table 1. Gene diagnostic strategies.
Genetic test

SignificanceFree DNA concentration

cfDNA concentration found higher in malignancies, other causes excluded.

Free DNA fragment length

This can help discriminate between the necrotic and apoptotic origin of cfDNA.

DNA methylation

DNA methylation in promoter region at CpG sites is an epigenetic tumor marker.

DNA mutation

DNA mutation is an important molecular marker for their role in malignant transformation.

Gene expression

Gene expression analysis is performed on cancer-related gene.

Figure 1. Demonstrate the release of CfDNA into the blood circulation by normal cells and cells involved in the pathogenic processes including cell
death. The human bloodstream contains cell-free DNA, cell-free RNA, and proteins that can be used as biomarkers for various diseases.

mutations) that block tumor suppressor gene functions ininhibiting cell proliferation, promoting DNA repair, and activating cell cycle checkpoints.14
Depending upon the technology, sensitivity of the method,
and analytical techniques used, these genetic cancer markers
can be detected in various types of samples from a patient’s
body, such as blood, urine, tissue, saliva, or a biopsy sample.
Minimally invasive detection of the tumor markers by the body
fluids (blood or urine) will also prove a useful method for thefollow-up monitoring of the therapeutic effects or prognosis of
the disease.15-17 Several gene test methods are used in HirahataInternational Cancer Clinic (HIC Clinic) and we find thesegenetic markers are high in their sensitivity and specificity forcancer detection. Table 1 shows various gene diagnosis strategies in molecular biology to detect the nucleic acid tumor
markers in a blood sample.

Free DNA concentration
Mandel and Metais discovered the presence of cfDNA in the
blood of healthy individuals in 1948.18,19 Decades later, many

researchers extended Mandel and Metais’ work and recognizedtumor-derived cfDNA (also known as circulating tumor
DNA-ctDNA) in the blood of cancer patients. It is mainly
found in the bloodstream; now it can be derived from cerebrospinal fluid and saliva. It can be excreted through urine withextremely low content.20,21 However, the amplifiable amount ofcirculating free DNA can be obtained from the blood plasmaof healthy and diseased individuals.22,23 Figure 1 represents thecharacteristic features of free DNA including its sources.
In general, cancer patients have an elevated level of circulating free DNA than healthy individuals.24-26 In 1977, scientists
made the significant observation of abnormally high levels of
cfDNA in plasma and serum from cancer patients compared tohealthy individuals.26,27 Previous studies showed that the concentration of cfDNA in cancer patients increased significantly
compared to a healthy individual that ranges from 0 to 1000 ng/
mL of blood, with an average of 180 ng/mL. In contrast,
cfDNA in healthy individuals ranges from 0 to 100 ng/mL ofblood, with an average of 30 ng/mL.26,28 The concentration offree circulating DNA in the plasma of cancer patients can be 3
to 10 times higher than the level of normal healthy samples ofQuraish et alplasma. The measurements and quantification of this free
DNA can be performed by Real-time quantitative PCR, digital PCR, fluorescent dye method, and other molecular biology
technologies.29-31 Circulating tumor DNA analysis can be used
as an early cancer screening test and can also be used for themonitoring of successful cancer treatment. For instance; adecrease in the quantity of circulating tumor DNA may suggest tumor size. Although the increased amounts of this circulating DNA (unless it is proved circulating tumor DNA
abbreviated as ctDNA) are not specific to cancer, it can be utilized as a screening method to indicate the need for further
complete biomarker assessment.32-34
Figure 2 represents the concept of free DNA concentration
among cancer patients and normal healthy individuals.

3

early diagnosis, and prognosis of cancer in the field of
oncology.36-39

DNA methylation
DNA methylation is an epigenetic mechanism involving theaddition of a methyl group at the fifth position of the pyrimidine ring of cytosine: that is, 5-methylcytosine Cytosine of
CpG islands at the promoter region (Figure 3). From previous

Free DNA fragment length
Another important feature of ctDNA is the large variation in
the fragment size. This characteristic can be referred to as thevariety of mechanisms that underlie dead cancer cells. In
healthy individuals, cfDNA is released predominantly throughapoptosis with small and uniform fragments of around 185 to
200 bp.35
In cancer patients, longer DNA fragments are considered toresult from necrotic cell death; necrosis creates a spectrum of
DNA fragments of various sizes due to incomplete and random digestion of genomic DNA by the nuclease enzyme,
interestingly; shorter fragments of ctDNA have been reported
in some tumor types (eg, hepatocellular carcinomas) as well aslarge fragments of cfDNA of thousands of base pairs.36-38
Circulating cell-free DNA concentration and fragment
length detection can be exploited for the prediction, prevention,

Figure 2. Diagrammatic representation of cfDNA concentration as abiomarker. The concentration is found higher in cancer patients than in
healthy individuals due to the presence of necrosis or apoptosis in the
body cells. Other causes of higher cfDNA concentration should be
excluded, such as pregnancy, transplant, and intense physical activity.35

Figure 3. Promoter methylation and gene silence. Promoter methylation (CH3 group addition on CpG islands) sometimes called hypermethylation, is an
important factor in carcinogenesis when it can prevent the proper expression (functions) of a tumor suppressor gene.

Cancer Informatics ﻿4research, it is obvious that DNA methylation plays many crucial roles in epigenetic regulation in cells, including gene
expression, cell proliferation, differentiation, genomic imprinting, X-chromosome inactivation, embryonic development, andcarcinogenesis.40,41 The mechanisms underlying cancer development are very complicated and the malignant transformation of a normal cell is forced by both epigenetic and genetic
alterations. It is also demonstrated that the initiation, maintenance, and progression of cancer involve epigenetic changes
including key processes of DNA methylation, histone modifications, nucleosome positioning, and aberrant expression of
non-coding RNAs, specifically microRNAs.42,43 Disruption in
epigenetic processes can lead to altered gene functions and cellular neoplastic transformation, therefore appropriate controlof DNA methylation is not only essential for regulating genetranscription but also plays a pivotal role in maintaining the
integrity of the genome and modulating immune response.41
Loss of proper maintenance of epigenetic control may result ininappropriate inhibition of the involved gene and various signaling pathways, leading to a disease state, such as cancer.
According to another report, aberrant DNA hypermethylation
when occurs in the CpG island in the promoter regions of specific transcription factors contributes to the formation and progression of tumors. Consequently, DNA methylation may be abiomarker of early cancer detection.44 Epigenetic modifications in the form of methylation within the promoter area may
stop the function of a gene and if the gene is a tumor suppressor gene, then this can lead to cancer generation. Therefore,
hypermethylation of the tumor suppressor gene can be a strong
progression marker of cancer.45,46 Several methods are in use
for the detection of methylation as a biomarker with theiradvantages and disadvantages. Some of the methods are costeffective but of medium or low sensitivity. Methylation-specific
PCR (MSPCR) is a sensitive method for the analysis of DNA
methylation patterns in CpG islands.47-49 The methylation
profile of DNA facilitates early diagnosis, prognosis, andscreening for cancer as reported in previous studies.44
Table 2 shows some of the important genes found methylated in various cancers.

DNA mutation
A mutation is a change in the nucleotide sequence of a genethat can result from errors in DNA replication during cell division, exposure to mutagens, or viral infection.56 With thedevelopment of new technologies for a more accurate understanding of the genome and potential gene therapies, thedetection of mutations in the cancer samples of blood and/or
tissue has an increasingly central role in terms of the genetic
diagnosis of cancer. It is a very important genetic marker andthe variation in sequence from normal can indicate the presence of a carcinogenic process.57-59 As a powerful technique inmolecular genetics, DNA sequencing provides analysis of genes

Table 2. Genes detected for methylation.
Gene

CancerP16

NSCLC, colon, bladder, esophageal, andstomach cancers

P15

AML, ALL, colon cancer, lung, and breast cancer

RAR-beta2

Colon, breast, and lung cancer

APC

Colon, gastric, and esophageal

DAPK

Lung cancer, lymph node metastasis

MLH1

Endometrial, uterine, colon, breast, and lungcancer

E-Cadherin

Thyroid carcinoma, breast, gastric, and livercancers

H-Cadherin

Lung cancer and ovarian cancers

RASSF1A

Lung cancer

MGMT

Brain tumor, colon cancer, lung and breast
cancer

BRCA1

Breast cancer and ovarian cancer

MAGE-A

Breast and esophageal cancer

GSTP

Prostate cancer

FHIT

Esophageal cancer

GATA

Lung cancer and esophageal cancer

DLEC1

Lung cancer

TSLC1

Lung cancer

NKEFB

Colorectal cancer

Involvement shows only the major data findings in our laboratory as well as inpublished articles.50-55

at the nucleotide level. The main focus of DNA sequencing is
to determine the sequence of small regions of interest (∼1 kilobase) using a PCR product as a template. DNA sequencing
could be used to check all small known and unknown DNA
variations; Dideoxynucleotide sequencing or Sanger sequencing represents the most widely used technique for sequencing
DNA.60 Among chain-termination methods, capillary electrophoresis is used (SeqStudio Genetic Analyzer, ThermoFisher
scientific) double-stranded DNA is denatured into singlestranded DNA with NaOH. A Sanger reaction consists of asingle-strand DNA, primer, and a mixture of a particular
ddNTP with normal dNTPs (eg, ddATP with dATP, dCTP,
dGTP, and dTTP). A fluorescent dye molecule is covalently
attached to the dideoxynucleotide. ddNTPs cannot form aphosphodiester bond with the next deoxynucleotide so theyterminate DNA chain elongation. This step is done in 4 separate reactions using a different ddNTP for each reaction.61 We
use advanced technology for the detection of mutations utilizing free circulating DNA and at present, we analyze the fullcoding sequence of the following genes mentioned in Table 3.

Quraish et al

5Gene Therapy for Cancer

Table 3. Genes sequenced for mutation test.
Names of the genes sequenced for mutation test

EGFR

APC

BRCA2

HRAS

p53

NRAS

BRAF

BRCA1

ALK

ERBB2

KRAS

JAK1

EGFR1

BCL2

ERBB4

FGFR1

FLT3

MAPK2

Oncogenic mutations found in the cancerous cell, are an
important nucleic acid tumor marker for cancer detection andprogress assessment during and after the treatment (Table 3).
Mutations can also be useful to determine the effectiveness andchemotherapy resistance.62,63

Gene expression
Altered gene expression (mostly hyper-expression of an oncogene) is a very common finding in nearly all types of cancers andcan be used as a molecular marker for the detection of cancerous
activity in a group of cells. We can detect the types of genes thatare either up or down-regulated in specific cancer types with theuse of microarray, real-time PCR, and digital PCR.
Microarray is useful for the mass analysis of oncogenes suchas to make a profile; this data provides a snapshot of all thetranscriptional activities in the biological sample. Expression
measurement analysis leads to an understanding of genes thatare being regulated under the disease conditions, including
cancer, both in basic medical science and clinical medicine.64
Cancer is one of the most dreaded genetic disorders, and it
develops either through acquired mutations or epigenetic
changes that lead to disruption in gene expressions of cancerous cells. Accordingly, microarray technology is allowing theconstruction of specific gene expression profiling that plays arole in the correct diagnosis of a specific cancer type. Gene
expression analysis also provides insight to understand theoncogenic pathways and discover novel biomarkers for clinicaldiagnosis.65 The power of these gene expression detection tools
has been applied to a wide range of applications such as discovering disease subtypes and identifying underlying mechanismsof disease or drug response.66,67 We analyze more than 100
oncogenes by the technology of our custom build microarray;
real-time quantitative PCR, digital PCR, and northern blot arealso used when required. RNA from cancer cells is utilized todetect the expression of oncogenes.
Table 4 shows the list of genes detected by microarray for
their expression.68-70
Genes listed in Table 4 are often found with altered expression in tumor samples. The expression can be detected by DNA
microarray, real-time quantitative PCR (qPCR), or digital
PCR; which is more sensitive than qPCR.

There are 3 types of standard therapies for the treatment ofcancer; chemotherapy, radiotherapy, and surgical treatment.
The prognosis of cancer with these treatments was never satisfactory and even with the remission achievement of 5 years;
some of the cancer cells or cancer-related molecular changes
remain undetected and can cause recurrence.
There are some other problems also associated with conventional therapies such as these conventional therapies are generally effective against early-stage localized tumors, whereas theireffectiveness is not the same against a later stage of cancer andthere are chances of frequent relapse after radical surgery andconventional therapies.71,72 Further, drug resistance appears to
be a serious problem in the treatment of cancer and a major
reason for chemotherapy failure in cancer.73 Moreover, various
agents used in conventional therapies are harmful both tonormal cells and cancer cells, which can lead to prominent side
effects.71,72 Another concern with the standard therapies is thequality of life of the patients during their remission or survival
period. While improving and alleviating the severity of symptoms, these conventional treatment methods adversely affectthe body and most of the time their side effects are systemic
due to their unspecific nature.74,75
Therefore, novel approaches have raised hope to significantly improve the survival rate of patients with cancers such as
immunotherapy, hormone therapy, gene therapy, and immunegene therapy. Currently, gene therapy approaches predominately practice in research laboratories. It has been recognized
as the capacity for genetic improvement through the correctionof the altered (mutated) gene or site-specific modifications thathave treatment as a target.72,76,77
Figure 4 demonstrates a summary of molecular gene therapy and immune-cellular gene therapy.
The main categories of antitumor immune-gene therapy
that can be customized according to the patient’s requirements
are given below, followed by a brief description of eachmethod.72,76,77 Table 5 summarizes the categories of genetherapy.

Immune cell therapy
Cancer has been identified as a leading cause of death worldwide. In recent years, cancer treatment has been revolutionizedwith the development of immunotherapies that aim to boost
the body’s immune system to more efficiently target anddestroy cancerous cells.78 Traditional therapies such as chemotherapy and radiotherapy cannot distinguish between normal
and cancerous cells, causing substantial toxicity to healthy tissue which can be a major limiting factor in their use, therefore,much attention is being paid to cancer immunotherapy which
is a personalized therapy that activates the intrinsic ability of
the immune system of the cancer patient through using thecomponent of the immune system to prevent and inverse theevasion activity by tumor, thus boost the immune system toCancer Informatics ﻿

6Table 4. Genes detected in expression analysis.
Names of the genes detected in expression analysis
Cyto-7

TNFSF10

SESN1

CEA

PSMA

NOTCH1

MAGE-A3 6

MDM2

MAPK1

PSA

VEGFR

FAS

DDIT3

CDKN1A

BRCA2

Bcl2

AKT1

Mucin-6

Mucin-12

S100

TRC3

PMS2

NOD1

Abl/Bcr

MCL1

MAP7

KIT

HPRT1

ESR1

CXCL1

Ki-67

BRCA1

BAX

AKAP13

VEGF-ATGFB1

RPL13A

RAB21

PGR

NLRP1

MMP2

MBD4

hk5

JUN

FOXA1

ERG

CTNNB1

mTOR

Bcr

ARAF

Topo-II

TP73

TERT

RORB

SCCA1

PGF

NFKB1

MMP13

MAS1

MAP2K5

ITGB3

C-FOS

ERCC1

CD38

CD40

BIRC5

AR

AFP

TP63

SRC

RB1

COX2

PALB2

MYC

MLH1

VEGFR3Cyto-20

IGFBP3

FLT1

Her-2

CFLAR

CCND1

BIRC2

APC

Ab1

p53

SOCS3

CD43

CD10

NSE

Mucin-1

c-Met

MAPK3

Cyto-19

IGF1R

FGF1

EGFR

HSP70

CASP3

BCL2L1ANGPT1

ABCG2

destroy cancer cell in the body.79 Immune cell therapy is a mostadvanced and entirely new paradigm in the field of cancer
treatment, different strategies such as immune cells, targeted
antibodies, cancer vaccines, adoptive cell transfer, cytokines,
tumor infecting viruses, and checkpoint inhibitors are used in
immunotherapy.80 It has been reported that various immune
cell types contribute toward developing innovative anticancer
strategies such as macrophages, neutrophils, NK cells, T-cells,and B-cells. The major form of cellular immunotherapies
includes T-cell-based, NK cell-based, and Dendritic cellbased.81,82 In this review, we describe briefly the major role
played by NK, NKT, and dendritic cells (DCs) and the mechanism used by these cells to get rid of cancer from the body.
Immune system cells do not recognize the tumor cells to
attack and as such, they cannot initiate an effective immune
response against tumor cells as they do for bacteria and viruses,
etc. In cancer, immune cells seem to be tolerant to the cancer
cells like their normal body cells.83,84 To remove this tolerance
and to make the immune cells activated against the tumor cells,
first, remove them from the patient’s body and then train andcultivate these cells ex vivo in a GMP-grade facility to makethem able to recognize the tumor cells. To produce clinicalgrade immune cells, such as NK, DC, and NKT, strategies aredeveloped to amplify these cells and modify them by transfection of these immune cells with cancer-recognizing elements
such as tumor antigens, immune cell receptors, and co-stimulatory molecules.84

NK cell therapy. NK cells are an important subunit of the
innate immune system that exhibits a potent tumor cytolytic
function against physiologically stressed cells such as tumor
cells and virally infected cells and induce an antigen-independent immune response.85,86 The function of NK cells is similar
to that of cytotoxic T-cells in the adaptive immune response ofvertebrates.87 Unlike the adaptive immune system, NK cells
exhibit tumor cytotoxicity against tumor and virus-infected
cells without prior sensitization via activation of NK cellactivating receptors against ligands present on tumor target
cells.88-90 As NK-mediated cytotoxicity develops in a TAAindependent (T cells target tumor-associated antigens) andMHC-unrestricted fashion, thus diverting the difficulty in
identifying defined tumor-specific antigens, and maintaining
the accurate distinction between normal and malignant
cells.91,92 Nk cells express several activating and inhibitory
receptors that recognize the altered expression of proteins on atarget cell and control the cytolytic function of NK cells. In
patients with cancer, NK cell function is inhibited due to the
reduced expression of NK cell-activating receptors, which
results in impairing their cytolytic activity. In this regard, adoptive immunotherapy with NK cells has emerged as a promisingsolution against several malignancies.93 Several approaches
have been developed to generate NK cells for adoptive immunotherapy. One of these approaches involves using cytokines,
such as IL-2, IL-12, IL-15, IL-18, and IL-21, to culture
and expand NK cells.94 These cytokines can upregulate theQuraish et al

7

Figure 4. In a broad sense, gene therapy involves restoring or manipulating the altered genes found in the diseased cells, as detected by the genetest or gene diagnosis. In the molecular gene therapy approach, a gene can be constructed and inserted in a mammalian expression plasmid andthen infused into the body to replace the function of a damaged or mutated gene, such as wild type TP53 gene or any other tumor suppressor gene(TSG). A plasmid can also be used to deliver the antigenic sequence of a tumor antigen (DNA Vaccine) which produces the peptide to present on thecell surface with a major histocompatibility complex (MHC) molecule to activate the cellular immunity against the tumor. Cytokines are another optionthat can be constructed in the plasmid to encode and modulate the immune system cells. Immuno-cellular gene therapy is an approach thatmodulates the immune system to work against cancerous cells; both humoral and cellular arms can be manipulated. Anti-tumor immune cells such as
NK, NKT, and DC can be activated and educated against the tumor in vivo by plasmid-based cytokines or DNA vaccines, these anti-tumor cells canalso be taken out of the body, expanded in vitro, educated by tumor peptide culture method or by transfection method and then re-infused into thebody.

Table 5. Major categories of gene therapy.
Category

Subcategories

Immune cell therapy

NK cell therapy
Dendritic cell therapy
NKT cell therapy

DNA vaccine therapy

Dendritic cell vaccines
Plasmid DNA vaccines

Cytokine therapy

IL-24 therapy
IL-12 therapy
IL-21 therapy

Tumor suppressor
gene therapy

P53 gene therapy
TRAIL gene therapy
FUS1 gene therapy

expression of NK cell-activating receptors present in NK cells,
thereby enhancing the anti-tumor activity of NK cells against
the cells that express the respective receptor ligands. Culturing
NK cells with growth-inactivated feeder cells may also be used

to enhance NK cell proliferation and activation. Various NK
cell sources are used for the adaptive immune system such as
autologous NK cells, allogeneic NK cells, NK cell lines fromperipheral blood and stem cells, and genetically engineered NK
cells.95,96
Dendritic cell therapy. Dendritic cells (DCs) represent a heterogeneous family of immune cells that act as a messenger between
the innate and adaptive immune systems and are critical for the
induction of protective immune responses against pathogens.97
These cells are considered the optimal antigen-presenting cells
that capture, process, and present antigenic material on major
histocompatibility complex (MHC) class I and II molecules tolymphocytes thus activating the adaptive immune response.97,98
Under resting conditions, DCs are inactive and in an immaturestate in blood circulation, once activated and mature theymigrate to secondary lymphoid tissues such as lymph nodes
where they interact with T-cells and B-cell to initiate adaptive
immune response.99,100 Maturation of DCs is crucial to transport antigens to tumor-associated draining lymph nodes for
initiation of T-cell activation which is characterized by upregulation of MHC class I and II, cytokines, and chemokines production that are necessary for activation of T-cell responses.101

Cancer Informatics ﻿

8

In DC-based immunotherapy, DC is obtained from the patientand modulated ex vivo to induce the immune system toward
tumor elimination.97 However, host immune systems involving
DCs are restricted in tumors due to several mechanisms,
including the low number of DCs in the tumor, reduced ability
for antigen presentation, and limited access to tumor antigens.
Recent advancements in cell culture techniques demonstratethat GM-CSF and IL-4 trigger monocytes in the peripheral
blood to induce significant activation of DCs. Therefore, artificially induced DCs can be administered intratumorally or
subcutaneously to effectively stimulate DC-mediated hostimmune responses.102,103
NKT cell therapy. NKT cells are another specialized subset of
lipid and glycolipid reactive T-lymphocyte that play an important role in immunosurveillance and antitumor immunity,
therefore they play an important role in the development ofeffective immunotherapy against cancer. These cells are foundin very small numbers in the peripheral blood and they sharethe molecular characteristics of both natural killer cells and T
cells.104,105 Unlike traditional T-cells that recognize peptide
antigens in the context of MHC I or II, NKT cells recognize
glycolipids presented within the context of the MHC I-likemolecule CD1d. These cell cells can be classified into 2 major
subsets: type I and type II NKT cells based on TCR rearrangements and glycolipid reactivity. Type I, invariant NKT (iNKT)
cells, are the well-characterized subset of CD1d-restricted
T-cells and express an invariant Vαβ TCR. Following antigen
recognition, iNKT cells rapidly secret cytokines and chemokines
which can influence both innate and adaptive immune systems.
Furthermore, activated NKT cells can directly induce cell deathin tumor cells and infected cells, therefore, play a critical role inautoimmune disease, infection, transplant immunology, andcancer.105-107 NKT cells can be engineered and cultivated tothose subpopulations of NKT cells ex vivo that are proven to be
most effective against cancer cells. They can also be made toactivate the adaptive T cell immunity in the body.108,109

DNA vaccine therapy
Cancer vaccines represent a promising strategy to induce a
specific and long-term immune response against tumor antigens (TAs). TAs are proteins overexpressed in tumor tissues
and play a critical role in tumor initiation, progression, andmetastasis.110,111 Cancer vaccines can be given to prevent thedevelopment of cancer in an otherwise healthy person and insuch a case these are called preventive vaccines, other vaccinesthat are given to treat cancer are called therapeutic vaccines.112
Vaccination can be cell-based such as dendritic cell vaccinespresenting tumor-associated antigen epitope or plasmid-basedwhich can be inoculated with or without liposome.
DNA Cancer Vaccine has been considered a very promising
immunotherapeutic approach to activate the immune response
against cancer due to its simplicity, stability, and safety. These

vaccines are based on the bacterial plasmid and contain genes
encoding tumor antigens coupled to CMV promotor.
There are optimally designed antitumor vaccination strategies are clinically tested and developed in the laboratory. These
vaccines can be delivered by a variety of different routes such as
intramuscular (IM), intradermal (ID), subcutaneous (SC), andmucosal.113,114 Once, a vaccine is delivered in the nucleus, theantigen encoded by the DNA vaccine is expressed and presented on major histocompatibility molecules (MHC) for T
cell activation.115
Dendritic cell vaccines. Dendritic cell (DC) vaccination is analternative form of immunotherapy and is a prime candidate toenrich the treatment possibilities for cancer. A dendritic cell
vaccine (sipuleucel-T) was first approved by FDA in 2010 andconsists of isolated PBMCS which is cultured with a GMCSF/Prostatic acid phosphate fusion. The use of sipuleucel-T
prolonged overall survival among men with metastatic castration-resistant prostate cancer.116 DC-based vaccination is anapproach to produce an immune response to eliminate tumor
cells and is safe, highly immunogenic, and able to activate ananti-tumor immune response. To generate the whole cell DC
vaccines, monocytes (immature cDCs) are isolated from thepatient’s peripheral blood. In the case of monocyte isolation,
immature moDCs are generated by culturing the isolated cells
in GM-CSF and IL-4. Once immature DCs are obtained, they
are activated ex vivo using a variety of cytokines and loaded withtumor-specific antigens. The matured DCs are then injectedback into the patient, usually via subcutaneous or intradermal
injections, although intravenous or direct injection into lymph
nodes, thus present antigens to activate cytotoxic T cells and canalso stimulate the B cells for antibody production, so DC vaccines can induce both innate and acquired immune systemcomponents. These vaccines are associated with limited toxicities, are therefore recognized as a relatively safe therapeutic
approach, and are being extensively evaluated in the clinic.117-119
Plasmid DNA vaccines. Plasmid DNA-based vaccines are
Third-Generation Vaccines. Fundamentally a non-viral vector(plasmid) is designed with a gene of interest (DNA or RNA), a
promoter, and antibiotic resistance gene and cloning sites. These
plasmid DNA vaccines can be administered into the bodythrough different routes, such as intramuscular, intravenous, orintradermal injection. A delivery vehicle is required to transport
the plasmid to the target cells. Non-viral Liposomal delivery
system is used to carry DNA vaccine plasmid to the cancer cells.
These vaccines are proven to be relatively specific and produce
high levels of transgene expression, as well as inducing no vector-related immune responses (Figure 5).120,121

Cytokine therapy
Cytokines such as interleukins and interferons are the key
regulator of the innate and adaptive immune system that has

Quraish et al

Figure 5. Basic components of a plasmid. A plasmid is constructed tohave a gene of interest (expression of which is required), origin of
replication (at which replication is initiated), multiple cloning sites(restriction sites for DNA insertion), antibiotic resistance gene (to selectthe transformed bacteria), and a promoter (transcription of an insertedgene).

therapeutic effects in various types of tumors they can be given
with or without other therapeutic agents.122,123 The function ofcytokines to activate the immune system in cancer was firststudied in a patient with metastatic melanoma and renal cancer. The interleukin-2 (IL-2) family of cytokines comprised of
IL-2, IL-7, IL-5, and IL-21 is the most targeted family ofcytokines in cancer immunotherapy,124 IFN-α was the firstcytokine approved for the treatment of human cancer. In recent
years, many cytokines, including interleukin (IL)-2, IL-12,
IL-15, IL-21, granulocyte-macrophage colony-stimulating
factor (GM-CSF), and interferon (IFN)-α have been evaluated in clinical trials for the immunotherapy of cancer.125
Cytokines are given with a non-viral delivery system such as
liposome and without liposome depending upon the requirement. These cytokines improve the overall reaction of theimmune system against the cancer cells and they can also stimulate NK cells, B-cells, and T-cells.122,123 The IL-2 family ofcytokines activates cytotoxic T-cells and natural killer cells
through the activation of STAT5 which is a transcription factor responsible for cellular differentiation.124

Tumor suppressor gene therapy
Tumor suppressor genes play a pivotal role in maintaining
genome integrity and regulating cell proliferation, cell-cycle
progression, differentiation, and crucial signaling functions
including apoptosis, autophagy, and necrosis. Tumor suppressor gene therapy has been observed to suppress tumor growth
via induction of apoptosis and cell cycle arrest.126 These genes
are inactivated by loss-of-function (LoF) mutations thatchange or block tumor suppressor genes’ normal functions.

9

Their loss-of-function mutations are related directly to thedevelopment of cancer. Therefore, the use of tumor suppressor genes as anticancer therapeutics has been investigated inboth experimental and clinical research, it has been reported
that therapeutic strategies targeting tumor suppressor genes
may help in preventing the progression of pancreaticcancer.127 Further, novel approaches including synthetic
lethality and collateral vulnerability screens have been developed to target gene defects in p53, PTEN, and BRCA1/2. In
addition, various studies have shown that a combination oftumor suppressor gene therapy with conventional cancer therapy can produce synergistic therapeutic benefits.128,129 Tumor
suppressor genes have been given intravenously with a targetspecific delivery system and can reduce cancer. Synthesis andconstruction of the complete coding sequence of wild-type
plasmids of various tumor suppressor genes are possible; thesetumor suppressor gene plasmids have various functions suchas apoptosis induction, cell cycle arrest, and anti-angiogenesis. All of these functions lead to the inhibition of tumor cell
growth.130

Challenges and future perspective

Cancer gene therapy involves the modification and manipulation of gene expression to cure cancer. Over the past few decades, it has developed as a potential and effective modality forthe treatment of cancer disease. Nevertheless, there are somechallenges including non-specific expression, low-efficiency
delivery, biosafety, and difficulty in utilizing newly discovered
potential genetic markers for clinical data and trials. Even
though, various novel genetic strategies have been developed tomake transgene expression and gene delivery safe and more
effective. However, there are still several issues that limited thesuccess of cancer gene therapy applications. Among them, thecrucial factors are targeting efficiency and safety. Recent
studies suggested that these issues could be resolved with thetechnique of targeted gene expression, including specificpromotor–operating expression and distinct delivery systems.
These specific promotors can be classified into 3 different categories: tissue-specific, cancer-specific, and tumor-specific.131,132
According to previous studies, cancer- and/or tumor-speciﬁc
promoters rather than tissue-specific could be a better choice to
avoid non-specific expression in a normal cell, as they can
restrict the expression of therapeutic genes in tumor cells,
meanwhile avoiding nonspecific expression and adverse effects
in normal cells.133 Moreover, the selection of an appropriate
tumor-specific promoter for a specific tumor is a critical factor
for the specificity, efficiency, and safety problems of target genetherapy in cancer.
In addition, the success of gene therapy in cancer treatment relies not only on a good genetic strategy but also
depends on a safe, efficient, and specific gene delivery system.
Accordingly, a wide variety of genetic vectors have beendeveloped for therapeutic gene delivery, among them, viralCancer Informatics ﻿10vectors have shown the highest potential for efficient genetherapy but have some limitations like immunogenicity, lessspecificity to the target cell, toxicity, insertion mutagenesis,
and limited genetic load, these limitations have encouraged
researchers to focus on the development of non-viral vectors,
supported by nanomedicines.72,134 Physicochemical characteristics of nanoparticles have developed a delivery systemthat utilizes passive, active, and stimuli-responsive targetingstrategies to improve drug delivery efficacy.135 Because
biosafety is the main concern in the design of gene delivery
systems. Ideally, a vector needs to have excellent biosafety,
biocompatibility, and low toxicity, as well as, should undergo
complete metabolization. Non-viral vectors have an important biosafety advantage over viral vectors. Among non-viral
vectors, nanostructure complexes called liposomes have been
considered to be promising vectorization strategies for genedelivery, which exhibit low toxicity, and non-immunogenicity
and are feasible to produce on a large scale.136,137 Althoughmany viral vectors are effective delivery vehicles for clinicalgene therapy, some are considered risky for oncogenesis.138,139
Therefore, further research is required with a focus on
biosafety risk management; risk assessment, and mitigationplans.140-143 Biosafety specialists play an important role indefining and meeting the need for an effective containment
framework, mitigation plan, well-managed gene therapy
protocol, biosafety strategies, and appropriate procedures inhandling gene therapy products for health care personnel.144
Another important obstacle in the way of successful cancer
gene therapy implementation is the hurdle in the utilizationof genetic markers in clinical practice. The large and expanding literature on cancer biomarkers asserts that these biomarkers can serve many clinical needs from risk stratification
to prognosis, diagnosis, screening, monitoring, drug toxicity
assessment, and prediction of therapeutic response, evenmany markers display promising results in the laboratory but
they are not translated from the academic research laboratory
in routine clinical care. Previous studies showed that insufficiency in sensitivity and specificity value makes them lessdesirable in clinical practice. Nevertheless, the new biomarkers are often clinically unfavorable and information provided
by the these biomarkers is either weak or not essential forclinical acceptance. Moreover, some biomarkers show reasonable sensitivity and specificity and can address a clear clinicalunmet need, but the strength of their predictive ability is
below a clinically defined threshold, therefore, not persuadeclinicians to adopt them. Another hindrance to biomarkers
reaching the clinic is the limited evidence about their performance in a clinical setting and inadequate systemic evaluationof the available evidence, or false discovery.145,146
To introduce genetic biomarkers to the clinic, it is indispensable to show a useful clinical application that is supported by
the validation data. The current cost of genetic tumor marker
testing is another factor related to their clinical application as it

is very costly to conduct a necessary trial to obtain approval
from the Food and Drug Administration (FDA).145
The Early Detection Research Network (EDRN), is anorganization with a mandate to discover, and validate cancer
biomarkers and introduce them to the clinic, so that they cancontribute to better patient care through the personalized
management of cancer.147 EDRN has implemented measures
to improve biomarker discovery and validation such as data
sharing, the use of common data elements, collaborative studies, and a strong emphasis on quality control. A well-designed
strategy to accomplish the mandate of the EDRN will lead todecreased incidences of false discovery as well as better usage offunding and time, by focusing improvement on well-validated
and high-performance biomarkers that will increase theirimplementation in the routine clinical care of cancer patients.
The use of careful study design, large datasets, continuous progression in genetic technologies and bioinformatics methodologies, better reporting, careful clinical validation, and large
collaborative studies facilitates the collection of valuable information about unmet clinical needs and improves the clinicalutility of biomarkers.145,146,148 Moreover, larger clinical trials areneeded to fully explore the potential of genetic tumor markers
for early diagnosis, prognosis, and drug selection. Professionals,
as well as researchers and scientists, play a crucial role and makeconcerted efforts to earn and safeguard public trust in clinical
trials and take a step to engage the public through current
discussions/forums, and provide them understandable information about gene drive technology.
Moreover, gene therapy will play a pivotal role in future cancer
care as part of multimodality treatment in combination with/or
following other forms of cancer therapies such as chemotherapy, radiation therapy, and surgery. It has been observed thattumor suppressor gene therapy asserts synergistic effects incombination with conventional therapy.129,149-151 Further
research should be focused on well-designed experiments incombinational therapy studies including gene therapy withchemotherapy, molecular inhibitors therapy, radiation therapy,
and immunotherapy will help in the development of costeffective therapeutic strategies to improve treatment outcomesfor cancer patients and decrease the death rate from cancer.

Conclusion

Gene therapy is one of the intriguing approaches in medical
genetics for the treatment of cancer which enables genetic
improvement through the correction of the defective (mutated)
gene or site-specific alteration that targets treatment. Cancer
gene therapy offers several potential treatment methods tofight this disease and has advantages over conventional therapies because high therapeutic doses can be administered via
gene therapy technique without systemic adverse effects.
Many of the past obstacles to treating cancer have been overcome with the use of gene therapy techniques and its potential
for precision medicine is worthwhile and its expectation forQuraish et al
curing and preventing cancer disease is promising. It has beenrecognized as the most emerging approach in the arena of biotechnology to the current status of progress as well as future
possibilities. Nevertheless, there is still a need for more rigorous
research in cancer genetics to improve the clinical utility ofemerging gene therapy technologies to ensure cancer elimination and normal cell maintenance. All scientists, pharmaceutical companies, and medical professionals must work together
to ensure that safe, life-long, and cost-effective gene therapies
become available to help treat patients with cancer.

Author Contributions

R.Q. and A.Q conceptualized, researched, and wrote thismanuscript. All authors have read and agreed to the publishedversion of the final manuscript.
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