Early detection of cancer in Nepal : Role of liquid biopsy

In this paper, we review population-based cancer screening programmes of different countries and share recommendations and relevant evidence for screening and early detection of common cancers in Nepal. The evidence-based recommendations provided in this Review are intended to act as a guide for policy makers, clinicians, and public health practitioners who are developing and implementing strategies in cancer control. We also discuss the role of liquid biopsy in early detection, diagnosis and monitoring of cancers using circulating biomarkers. Despite challenges, time has come to include cell free circulating tumor DNA and circulating tumor cells, as a parameters for early detection of cancer in the days to come. ABSTRACT Sameer Chhetri Aryal1, Gopi Aryal2


INTRODUCTION
Cancer is a molecular disease associated with alterations in the genome.Early diagnosis identifies symptomatic cancer cases at the earliest possible stage.Delays in cancer detection and late stage presentation are common problems in developing countries like Nepal.Even in countries with strong health systems and services, many cancer cases are diagnosed at a late-stage.The consequences of delayed or inaccessible cancer care are lower likelihood of survival, greater morbidity and higher costs of care.
Screening-seeks asymptomatic cancer or pre-cancerous lesions in a target population.There is no National cancerscreening program in Nepal till date.One of the most troubling misconceptions is the lack of belief people have in using routine diagnostic procedures to detect cancers while they are still in a manageable state.Many of these individuals believe that they are too young or that the screening tests are too expensive for serious consideration.

Keywords:
Cancer; Liquid biopsy; Detection; Nepal; Circulating Tumor DNA; Screening; Over the ten-year period from 2003 to 2012, the top ten cancers in Nepal are given in Table 1. 1 Predisposing factors for cancers:

MOLECULAR BASIS OF CANCER
Development of all human cancer requires inherited germ line mutation or acquired mutation.Most important mutation involves proto-oncogenes, tumor suppressor gene and gene regulating apoptosis.DNA viruses such as papillomaviruses can promote the development of cancer by sequestering the products of tumor suppressor genes-in particular, the Rb protein, which regulates cell division, and the p53 protein, which is thought to act as an emergency brake on cell division in cells that have suffered genetic damage and to call a halt to cell division in senescent cells with shortened telomeres.
Cancer is the end-result of the accumulation of cell divisions in stem cells.However, cell division can lead to a variety of cancer-promoting errors, such as mutations and epigenetic mistakes occurring during DNA replication, chromosome aberrations arising during mitosis, errors in the distribution of cell-fate determinants between the daughter cells, and failures to restore physical interactions with other tissue components.As carcinogenesis progresses, the accumulation of DNA errors promotes cell division and eventually triggers cell division under permissive extracellular environments.The accumulation of cell divisions in stem cells drives not only the accumulation of the DNA alterations required for carcinogenesis, but also the formation and growth of the abnormal cell populations that characterize the disease. 2

CANCER SCREENING
According to the recommendation by the Council of the European Union 3 , it is an ethical, legal and social prerequisite that cancer screening should only be offered to fully informed people with no symptoms if the screening is proved to decrease disease-specific mortality, if the benefits and risks are well known and if the cost-effectiveness of screening is acceptable.
The balance of benefits and harms is a strongly debated topic, particularly in the field of population-based cancer screening.The benefits from cancer screening are reduced cancer death, decreased chance of advanced disease at diagnosis and less expensive treatment.The harms include over-diagnosis, overtreatment, false-positive and false negative findings, Each year, the American Cancer Society (ACS) publishes a summary of its guidelines for early cancer detection, data and trends in cancer screening rates from the National Health Interview Survey, and select issues related to cancer screening (Table 2). 4

LUNG CANCER SCREENING
The ACS recommends annual screening for lung cancer with LDCT in adults aged 55 to 74 years in relatively good health who: Screening decisions should be individualized, based on patient preferences, life expectancy, health status, and prior screening history.If a decision is made to continue screening patient should be offered options as listed above.
Individuals should be discouraged from continuing screening.

Endometrial Women, at menopause Stomach
At the time of menopause, women should be informed about risks and symptoms of endometrial cancer and strongly encouraged to report any unexpected bleeding or spotting to their physicians

Current or former smokers ( 55-74 years ) in good health with at least a 30 pack/year
Low-dose helical CT Annual screening in adults who: l currently smoke or have quit within the past 15 years; and l have at least a 30 pack-year smoking history; and l receive evidence-based smoking cessation counseling, if they are current smokers; and l have undergone a process of informed/shared decision making that included in formation about the potential benefits, limitations, and harms of screening with low-dose CT; and l have access to a high-volume, high quality lung cancer screening and treatment center.

Prostate Men ≥50 years
Prostate specific antigen test (PSA) with or without digital rectal examination (DRE) Men who have at least a 10-years life expectancy should have an opportunity to make an informed decision with their health care provider about whether to be screened for prostate cancer after receiving information about the potential benefits, risks, and uncertainties associated with prostate cancer screening; prostate cancer screening should not occur without an informed decision-making process • Currently smoke or have quit within the past 15 years; and • Have at least a 30-pack-year smoking history; and • Receive evidence-based smoking-cessation counseling, if they are current smokers; and • Have undergone a process of informed/shared decision making that included information about the potential benefits, limitations, and harms of screening with LDCT and • Have access to a high-volume, high-quality lung cancer screening and treatment center.

GASTRIC CANCER SCREENING
The following methods were assessed for gastric cancer screening: I. Upper gastrointestinal series (radiographic screening) II.Gastrointestinal endoscopy (endoscopic screening)

III. Helicobacter pylori antibody test and serum pepsinogen tests
Analytic framework for gastric cancer screening is shown in figure 1. 5

SCREENING FOR BREAST CANCER
The ACS recommends that women aged 45 to 54 years and those aged 40 to 44 years who choose to begin screening before age 45, should be screened annually, and women aged 55 years and older should transition to biennial screening or can continue annual screening if that is their preference.

SCREENING WOMEN AT HIGH RISK
Annual screening mammography and magnetic resonance imaging (MRI) starting at age 30 years are recommended for women with a known BRCA mutation, women who are untested but have a first-degree relative with a BRCA mutation, or women with an approximately 20% to 25% or greater lifetime risk of breast cancer based upon specialized breast cancer risk-estimation models capable of pedigree analysis of first-degree and second-degree relatives on both the maternal and paternal sides.Annual MRI and mammography also are recommended for women who were treated for Hodgkin disease with radiation to the chest between ages 10 and 30 years and women with Li-Fraumeni, Cowden, and Bannayan-Riley-Ruvalcaba syndromes.Analytical framework for breast cancer is shown in figure 2. 6

Special Considerations
These recommendations are intended for women at average risk and do not apply to women with a history of cervical cancer; women who were exposed in utero to diethylstilbestrol; women who are immunocompromised by organ transplantation, chemotherapy, or chronic DOI : 10.3126/jpn.v%vi%i.20893corticosteroid treatment; or women who are positive for the human immunodeficiency virus.In addition, women who have had their cervix removed should not be screened unless they have a history of CIN2 or a more severe diagnosis.Women who have undergone a subtotal (supracervical) hysterectomy should be screened following the recommendations for average-risk women who have not undergone a hysterectomy.

Vaccination Against HPV
The ACS recommends vaccination of all children at ages 11 and 12 years to protect against HPV infections that lead to several cancers and pre-cancers.

Figure 2: (1) Compared with no screening (or other screening strategies), is there direct evidence that the mortality from breast cancer is reduced with the following screening methods? (a) Clinical breast examination (CBE). (b) Mammography (MMG). (c) Mammography with clinical breast examination (MMG + CBE). (d) Ultrasonography with and without mammography (US ± MMG). To appropriately determinate the level of evidence, the primary outcomes of mortality from breast cancer must be ascertained. (2) What age group was the preferable target for breast cancer screening? (3) Can the screening test accurately detect breast cancer? What are the sensitivity and specificity rates of the screening test? (4) What are the potential harms of screening tests, and how often do they occur? (5) Can the diagnostic test accurately detect the target women? (6) What are the potential harms of diagnostic tests, and how often do they occur? 7) (For breast cancer patients, how are the efficacy and effectiveness of treatment?
A liquid biopsy is an analysis of liquid biomarkers that can be isolated from body fluids (urine, feces, saliva, sputum, pleural fluid, and cerebrospinal fluid) and blood.Liquid biopsies, mainly represented by circulating tumor cells, circulating tumor DNA, tumor exosomes, and microRNAs, have the potential to assess various biomarkers for early detection of cancer, monitoring tumor progression along with prognosis.
Biomarkers that are currently in use or under investigation in liquid biopsies are also shown in the   4) Does screening result in adverse effects compared to no screening?( 5) Can the diagnostic test accurately detect the target cancer?The diagnostic method is LEEP (loop electrosurgical excision procedure).( 6) Does the diagnostic test result in adverse effects compared to no test?(7) For cervical cancer patients, does any treatment reduce the incidence of an intermediate outcome compared to no treatment (or other treatment)?( 8) Does any treatment result in adverse effects?
exosomes.These circulating biomarkers are applied to guide initial diagnosis and treatment monitoring for patients with pancreatic cancer. 9mor-educated blood platelets (TEP) are involved in the progression and spread of several solid tumors, and spliced TEP RNA surrogate signatures can provide specific information on the presence, location, and molecular characteristics of cancers.TEP samples from patients with lung, brain, and breast cancers, have been tested, and it has been shown that TEPs from patients with cancer are distinct from those with inflammatory and other noncancerous diseases.Ultimately, TEP RNA may complement currently used biomolecules employed for liquid biopsy diagnosis, potentially enhancing the detection of cancer in an early stage and facilitating noninvasive disease monitoring. 10erging evidence has demonstrated the feasibility of circulating miRNAs as robust non-invasive biomarkers for the diagnosis in colorectal cancer.The use of circulating miRNAs for the early detection of colorectal cancer (CRC) is of particular interest as it can offer a potential complementary approach to screening colonoscopy. 10ll free circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), exosomes and tumor-educated platelets (TEPs), have also been widely investigated for their potential role in lung cancer diagnosis. 11antitative real-time polymerase chain reaction data showed circulating miRNA-22-3p had significantly different expression in patients with precancerous lesions or gastric adenocarcinoma. 12rrent available imaging modalities do not reveal micrometastasis and tumor biopsy is an invasive method to detect early stage or recurrent cancer, signifying the need for an inexpensive, non-invasive diagnostic modality.Current evidence suggests that increasing levels of ctDNA in breast cancer can be of significant diagnostic value for early detection of breast cancer. 13inary cfNA holds potential for a more sensitive alternative to blood biopsies and urine sediment-based tests for clinical use in GU cancers. 14

DISCUSSION
Lack of knowledge and awareness about cancer, its risk factors and negligence of the early warning signs play crucial role in raising the incidence of the disease.Awareness programs through newspapers, radio, television and health campaigns regarding the early signs of cancer and benefit of detection at early stage are important factors to reduce cancer mortality.Three is significant challenges for Cancer Screening.For instance, high false-positive rates from mammography in breast cancer screening, low-dose CT in lung cancer screening, and prostate-specific antigen (PSA) screening represent a significant cost to the healthcare system, with resulting mental and physical morbidity.
Early signs of cancer In Nepal, the major cancers in males are lung cancer followed by stomach cancer.Among females, the three common cancers are cervix followed by breast and lung. 1 The increase incidence of cancer is largely attributable to a multitude of factors including an ageing population, increased life expectancy, a high prevalence of modifiable risk factors, poorly integrated primary and secondary prevention programmes, and inefficient or inaccessible treatment delivery systems. 15yal G et al In 2017, WHO member states adopted the draft resolution, which underscores cancer control as a national public health priority, with specific emphasis on screening and assessment of screening programmes. 16llowing are the six essential elements of national cancer screening programs: 1. Policy elements  The attitude of pathologists to and level of involvement in the practice of liquid biopsies, including mastering the methods employed in molecular analysis of blood samples, need close attention.Regardless of the level of involvement of pathologists in this new field, it is mandatory that oncologists, radiologist and pathologists work together to coordinate the pre-analytical, analytical, and post-analytical phases of molecular assessment of tissue and liquid samples of individual cancer patients.

The challenges include:
(1) Implementation of effective and efficient procedures for reception and analysis of liquid and tissue samples for histopathological and molecular evaluation and (2) Assuring short turn-around times (TAT) to facilitate rapid optimization of individual patient treatment. 18

CONCLUSION
Regardless of income levels, countries around the world are identifying approaches to offer cancer-screening DOI : 10.3126/jpn.v%vi%i.20893programmes to their residents via financing, community engagement, and strengthening of health-care systems.By recognizing that screening is a step in the continuum of cancer care, governments can use available technologies and human resources to help detect cancers early and treat them successfully.
Early detection of cancer and screening programmes across Nepal including a well-developed analytical framework need to develop.Periodic follow-up and quality assurance is essential for the same.Despite challenges, time has come to include cell free circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), as a parameters for early detection of cancer in the days to come.

Figure 1 :
Figure 1: Analytic framework and key questions for gastric cancer screening.1.Is there direct evidence that the mortality from gastric cancer is reduced with the following screening methods?(a) Upper gastrointestinal series (UGI), (b) gastrointestinal endoscopy (GFS), (c) serum pepsinogen test (PG) Helicobacter pylori antibody (HP), (d) Helicobacter pylori antibody (HP), (e) A combined method of Helicobacter pylori antibody and serum pepsinogen test (HP + PG). 2. Can the screening test accurately detect gastric cancer?What are the sensitivity and specificity of the screening test? 3. What are the potential harms of the screening tests, and how often do they occur?4. Can the diagnostic test accurately diagnose gastric cancers? 5. What are the potential harms of the diagnostic examination, and how often do they occur?6.For gastric cancer patients, how are the efficacy and effectiveness of the treatment?7. What are the potential harms of gastric cancers treatment, and how often do they occur?

Figure 3 :
Figure 3: (1) is there direct evidence that following screening, the incidence and/or mortality are reduced?(2) Is the prevalence of cervical cancer in the target group?What strategy can reliably identify a high-risk group from among averagerisk persons?(3) Can the screening test accurately detect the target cancer?The screening methods are conventional cytology, liquid-based cytology, combination of HPV testing and cytology and HPV testing alone.(4) Does screening result in adverse effects compared to no screening?(5)Can the diagnostic test accurately detect the target cancer?The diagnostic method is LEEP (loop electrosurgical excision procedure).(6) Does the diagnostic test result in adverse effects compared to no test?(7) For cervical cancer patients, does any treatment reduce the incidence of an intermediate outcome compared to no treatment (or other treatment)?(8) Does any treatment result in adverse effects?

3. Development of a strong infrastructure for screening implementation 4 . 5 . Planning for community engagement 6 .
Establishment of Monitoring and assessment system Implementation science researchThere is no national or provincial cancer-screening program in Nepal.As a country with limited resources, Nepalese government need to start population based cancer-screening program at least for cancer of lung, stomach, cervix, breast and bowel.We have proposed organogram of cancer screening program in Nepal (fig.4).The advent of therapies targeting genomic alterations has improved the care of patients with certain types of cancer.While molecular targets were initially detected in nucleic acid samples extracted from tumor tissue, detection of nucleic acids in circulating blood has allowed the development of what has become known as liquid biopsies.

Figure 4 :
Figure 4: Proposed organogram for cancer screening program in Nepal.

Colorectal Men and women, aged ≥ 50 years , for all tests listed
Adults aged 45 years and older should undergo regular screening with either a high-sensitivity stool-based test or a structural (visual) exam, depending on patient preference and test availability.As part of the screening process, all positive results on non-colonoscopy screening tests should be followed up with timely colonoscopy.Adults in good health with a life expectancy of greater than 10 y should continue screening through the age of 75 years .
The vaccination series can be started beginning at age 9 years.

Table 3 . Comparison of different cancer detection methods and their clinical utilities
DOI : 10.3126/jpn.v%vi%i.20893

Table 3 . Comparison of different cancer detection methods and their clinical utilities ( continued )
DOI : 10.3126/jpn.v%vi%i.20893