In both developed and developing countries, cancer is a major health issue and a leading cause of mortality that is still on the increase, worldwide. According to the International Agency for Research on Cancer, in 2018, 9.6 million individuals died from cancer, an increase from 8.2 million in 2012 and 7.6 million in 2008 (1-3). Tumorigenesis is a multi-step, multi-factorial disease described by genetic and epigenetic changes, which is difficult to control and prevent (4). In 2012, the WHO's International Agency for Research on Cancer predicted that by 2030, worldwide, there would be 21.7 million newly diagnosed cancer cases and 13 million cancer deaths as a result of population growth and the increase in life expectancy (5).

Cancer is a major health problem that affects individuals globally. Several types of cancer can be avoided if diagnosed early enough. However, tumors such as lung, colon, and breast cancers frequently have late-stage diagnosis. Despite efforts to ensure survival is prolonged, only a moderate improvement has been achieved in cancer patients. Failure to diagnose cancer early generally leads to ineffective treatment and an even worse prognosis. The availability of robust diagnostic biomarkers is critical for diagnosing cancer patients at an early stage and thereby greatly reducing overall mortality rates. (6).

Circulating molecular biomarkers have increasingly been used as a liquid biopsy in the peripheral blood and have the benefit of being easily accessible, with early detection, and reproducibility (7). Circulating tumor cells, circulating DNA, and microRNAs have been studied as a detection tool and prognosis of various cancer types (8-13).

DNA is a molecule that may be found inside and outside of cells. Extracellular DNA can be found in blood and other body fluids. Cell-free DNA refers to the degraded DNA fragments floating in the circulation (cfDNA). DNA in the bloodstream releases apoptotic or necrotic cells. The length of DNA fragments and distribution of DNA size could signify cfDNA source (14). Apoptosis of the cell naturally occurs, and DNA is divided into similar fragments of 185-200 bp. However, tumor necrosis produces similar fragments of DNA in variable lengths generally >200 bp (15). Circulating tumor DNAs (ctDNAs) based DNA integrity index served as a possible indicator of prognosis in hepatocellular carcinoma, lymphoma, colorectal, lung, and breast cancer (15-19). DNA analysis can be conducted on the basis of ctDNA (from a liquid biopsy) as well as directly isolated DNA from tumor tissue acquired by biopsy or excision (20).

According to Iqbal et al (21) presence of cfDNA in blood, although reported in 1948 by Mandel and Metais (22), was rediscovered after 30 years in autoimmune disorders by Tan et al in 1966(23) and in cancer by Leon et al in 1977(24).

Apoptosis is the source of cfDNA in a healthy person, raising shorter and evenly sized DNA fragments. Furthermore, in cancer, necrosis results in unequal longer DNA fragments in addition to the shorter apoptotic fragment (25-27). As a result, higher levels of longer DNA fragments in the blood have been identified as a useful indicator of the existence of malignant tumor DNA (26-28). The cfDNA concentration in serum is higher in patients with cancer when compared to healthy individuals (5,29-31).

The variability of cfDNA levels in patients is most probably associated with tumor stage, burden, cellular turnover, vascularity, and response to therapy with the highest levels reported in patients with metastatic and advanced disease (32).

cfDNA levels have been found to be elevated in a variety of cancers (7). One measure of cfDNA fragmentation is cfDNA integrity (cfDI), which is calculated as the ratio of longer to shorter DNA fragment concentrations at the same genetic location (29).

A liquid biopsy is a viable alternative consisting of the circulating analysis (cfDI). This main advantage of this method is that it is less invasive, using just a sample of peripheral blood. During the past two decades, cfDI analysis has emerged as a promising tool for cancer diagnosis and prognosis (33,34).

The Arthrobacter luteus (ALU) repeats are the most predominant repetitive sequences in the human genome, 300 bp in length, with 1.4x10⁶ copy number per genome. Most studies used DNA integrity, defined as the ratio of ALU 247 long fragments released from necrotic cells and ALU 115 short fragments released from normal cells (35).

ALU-quantitative PCR (qPCR) has become the most widely used technology for detecting the DNA integrity index (32). ALU covers over 10% of the human genome (36). Research has been conducted to assess the potential use of cfDI from the ALU variable as a diagnostic biomarker for a variety of cancers, such as breast and prostate cancer (37).

A higher portion of longer DNA fragments has been recommended as a cancer detection biomarker (26). Several formulae have been presented to objectively calculate the ‘DNA integrity index’ as a ratio of longer and smaller fragments. Umetani et al (27,38) determined the pure ratio of ALU 247 and ALU 115 concentrations in patients' blood, while Wang et al (39) assessed DNA integrity in patient plasma using a calculation based on delta-Cp values. Patients with ovarian, breast and colorectal cancer had higher DNA integrities in serum and plasma than controls, according to both authors (27,38). Other studies, on the other hand, could not find a difference in DNA integrity values in the same tumor types (40-42).

However, in different studies, the performance of ALU repeat as a biomarker for cancer diagnosis varied widely. Therefore, this systematic study, to the best of our knowledge, is the first to clarify the diagnostic and prognostic role of ALU elements as a molecular marker of cancer.

Cancer is a major global health problem due to the increasing incidence and fatality rates. An early cancer diagnosis is crucial as it can improve the chances of survival for cancer patients and decrease the mortality rate. At present, liquid biopsies are promising due to their potential advantages, including reliability, easy access, and reproducibility (11).

In the present review, several studies supported the use of liquid biopsy in cancer as being innovative. To determine whether this liquid biopsy could assist the diagnostic or assessment of treatment response, 36 articles were included to identify the ALU sequences as a biomarker in cancer. The limitations of data retrieved are mostly related to the 36 articles as there is great heterogeneity in studies, it is difficult to analyse the subgroup study, the articles constitute a small sample size, little research highlighting the differences of ALU and cfDI at different stages is available, and the cut-off values vary widely between studies and were missing in other studies.

The present review identified the role of ALU element in cancer progression. Collectively, our data indicated that ALU elements can be used as a biomarker (29,35,44,47,49,52,54,63). The use of cfDNA, for early diagnosis, prognosis biomarkers and monitoring of therapy have been a significant advancement in clinical medicine (18,47,51,52,60-62).

As mentioned previously, not all studies have confirmed that ALU levels vary with tumor development and progression, which may elucidate that cancer is a heterogeneous disease.

cfDI was subsequently evaluated for its usefulness in cancer diagnosis and prognosis (5,15,31,32,34,48,58). Higher cfDI values in cancer patients vs. healthy controls were identified in many studies (31,34,57,58,61). By contrast, lower cfDI was observed in different studies; however, some articles with a focus on metastatic breast cancer (10,45), recurrent breast cancer (46), or first cycle of vaccination (53,62) were few and inconsistent.

There are few studies highlight of the ALU 260/111 in cancer (10,18,29,46) so is not possible to determinate the role of it as biomarker.

Several studies have identified an altered cfDI in patients compared to controls. However, these studies are heterogeneous, some studies showed a reduced cfDI in patients, while others reported an increased cfDI. Various hypotheses have been posited to understand the underlying reason. cfDNA from healthy individuals had 3- to 5-fold multiples of nucleosome-associated DNA length, and longer fragments than cfDNA from pancreatic cancer patients using direct visualization by gel electrophoresis (64). Jiang et al (64) found increased levels of shorter mitochondrial DNA molecules in the plasma of cancer patients compared to healthy people using massively parallel sequencing.

The present study included 36 studies with many discrepancies. This was due to the fact that they were highly heterogeneous studies and not all studies included different stages. In addition, not enough data were available for a specific cancer type and occasionally subjects were limited, there were differences in molecular methods used and source of samples could influence the results. This heterogeneity may affect clarification and whether ALU can act as a biomarker in cancer disease. Thus, further studies are needed to better clarify the role of the ALU element in specific subgroups.

In general, the results obtained from the present systematic review show that the expression of ALU-247 and ALU-115, and cfDI concentration is a rising trend associated with cancer and cancer stage. As a result, circulating cfDNA may be significantly related to tumor cell turnover and tumor progression, indicating biologic tumor aggressiveness. Thus, the circulating cfDI could be suitable for monitoring cancer progression. In addition, large and multicenter sample groups must be studied to corroborate the findings.

Liquid biopsy is becoming the focus of future tumor diagnosis and treatment research. It is less invasive and painful, can be collected in a short period of time, can be collected regardless of where the target organ is located, can be repeated continually, and can represent cancer volume in real-time. However, there are disadvantages, such as if the amount of cfDNA is insufficient, correct information may not be collected. Thus, analytical technology must be established, and the patient's condition or underlying disease may influence the results. Liquid biopsies, as with any other test method, involve large cohort studies to determine their efficacy and sensitivity to various disorders.

The limitation of the present study was mainly to mention the results of ALU and cfDI in blood samples without mentioning the in vitro information regarding ALU and cfDI. This aspect should be investigated in future work.

In summary, findings of the review suggest that cfDI can be a significant predictor of developing cancer in patients and could be a useful marker in a molecular, blood-based multi-marker assay.

The current systematic review assessed the total ALU sequence levels and its index are promising biomarkers for the purpose of investigation or prognosis of cancer. However, because of the heterogeneity between studies, the difference of ALU value in different cancers (type or stage) therefore makes it difficult to compare between different types of cancer. Further studies and meta-analyses are needed for the final conclusion to explore the diagnostic role of ALU in malignant diseases, especially when combined with other cancer biomarkers.