Circulating nucleic acids in plasma and serum (CNAPS): applications in oncology

Integrity of the DNA strand

Using PCR, Wang et al⁵⁷ found that long DNA fragments, related to necrosis phenomena, could be distinguished from shorter fragments, produced by physiological apoptosis phenomena. These findings gave rise to the so-called integrity index, based on the ratio between long and short cfDNA fragments. The integrity index was established based on gynecologic and breast cancers,⁵⁷ with the results obtained from 61 patients compared with those from 65 patients without neoplastic disease. It was found that the area under the curve for the DNA integrity index was 0.911 in cancer patients in relation to patients without cancer, with 100% sensitivity and 62% specificity. The authors suggested that the integrity index provides a simple and inexpensive way to detect cancer.⁵⁷

A number of transposable elements of the genome, such as ALU and LINE1 sequences, can be easily detected and have been associated with tumor necrosis. Specifically, ALU sequences are short elements (typically 300 nucleotides in length) that account for more than 10% of the human genome. Umetani et al developed a method to measure the integrity of cfDNA in serum using quantitative PCR for ALU repeats, and suggested that the integrity index is increased at the early stages of the disease and a promising molecular biomarker for detecting colorectal and breast cancer.^58,59 Moreover, it might serve as a sensitive method to detect nodal metastases in the early stages, having an even higher predictive value than the clinicopathological variables commonly used, such as tumor size and stage.⁵⁹

In a recent study in patients with hepatitis B virus-related hepatocellular carcinoma, the integrity index was associated with tumor size, TNM Classification of Malignant Tumors (TMN) stage, and nodal metastasis, and showed a more discriminatory power than total cfDNA concentration.⁶⁰

Further, in other recent studies, the clinical value of the integrity index has been demonstrated, not only for the diagnosis but also the monitoring of the disease. For example, it has been found that the integrity index decreases in parallel to the response to surgical treatments for head and neck tumors⁶¹ and melanoma,⁶² to chemotherapy in leukemia,⁶³ to radiotherapy in nasopharyngeal tumors,⁶⁴ and to chemoradiotherapy in colorectal cancer.⁵² Moreover, it has been reported that patients with high concentrations of integrity DNA had shorter disease-free intervals.⁶⁴

Alterations in the integrity of DNA have also been detected in other organic liquids, such as urine, and can be used in the early diagnosis of bladder cancer without cytology. ⁶⁵ Some authors have emphasized that the integrity index meets many of the requirements of a universal biomarker;¹⁰ however, other studies have not supported this idea.⁶⁶

Gene mutations

As previously mentioned, many studies have shown that gene mutations found in primary tumors can be also detected in cfDNA circulating in plasma. The literature reports a wide percentage range of patients in whose plasma such mutations have been detected. The differences between studies might be due to technical reasons, since sensitive techniques are needed to detect very small amounts of mutated DNA among larger quantities of un-mutated DNA, which has a dilution effect.

The gene that has most frequently been examined is the KRAS oncogene, which is mutated in variable rates in high-incidence tumors, such as colon, pancreas, lung and thyroid tumors,⁹ and has intrinsic characteristics that make it useful as a marker.⁴⁹ In fact, the prevalence of KRAS mutations is near 50%. Such mutations occur in the early stages of carcinogenesis and are mostly produced at specific sites (particularly in codon 12), which facilitate its detection using PCR-based techniques.

With respect to colorectal cancer, KRAS mutations have been found in 40%–50% of patients, and have also been detected in the plasma or serum of 25%–30% of patients.⁴⁹ Kopreski et al⁶⁷ tried to correlate the detection of KRAS mutations in plasma with clinicopathological findings detected by colonoscopy. They enrolled into their study one of the largest series of colorectal cancer patients that has ever been enrolled into similar studies, and detected KRAS mutations in plasma from 83% of patients whose tumors had such mutations.⁶⁷ Moreover, they also found that some patients with apparently normal colonoscopy had KRAS mutations in plasma, which might be related to the presence of precancer lesions, thus suggesting the technique’s suitability for early diagnosis and screening.⁶⁷

In a review by Sorenson,⁶⁸ the author defended the detection of KRAS mutations in plasma as a specific marker for gastrointestinal tumors, with 2.5% false positives, although the review of studies showed that the concordance with KRAS plasmatic mutations with respect to primary tumor was only 50%. It has also been reported that other mutations, different from those in primary tumors, can be detected in plasma from cancer patients, which might be due to clone heterogeneity in tumors.⁶⁹

In patients with pancreas cancer, it has been shown that detecting KRAS mutations in plasma is useful for early diagnosis as a complementary marker together with other serum markers (eg, CA19.9).⁷⁰ Other researchers have also used it for disease monitoring.⁷¹

In a prospective study conducted by Gormally et al⁷² of a healthy population, KRAS mutations were detected in plasma from 1.2% subjects and p53 mutations in 3.6%. The authors suggested that KRAS mutations are detectable in plasma before the diagnosis of bladder cancer. In contrast, KRAS mutations have been detected by other researchers in up to 30% of healthy individuals;⁹ this has been interpreted as a limitation of the diagnosis and screening value.

With respect to mutations in other genes, Chen et al⁷³ used an ultrasensitive technique to detect p53 mutations in plasma from patients with Stage II and III breast cancer, and their results showed a correlation between such detection and the clinical course of the disease after therapy.

The value of detection of tumor mutations in plasma for monitoring the response to treatment has been analyzed in several studies. Diehl et al⁷⁴ showed that the detection and quantification of mutated cfDNA in plasma from colon cancer patients undergoing surgery and chemotherapy were more useful for monitoring than the quantitation of CEA in serum. In addition, a recent study has shown that the use of panels with the most frequent mutations in colorectal cancer (APC, KRAS, TP53, PIK3CA, and BRAF) was more useful than using CEA and CA19.9 levels.⁷⁵ Further, it has been suggested that, in the postoperative period, the levels of mutated KRAS in plasma are a more powerful predictor of recurrence than Dukes stage.⁷⁶

In breast cancer patients, the presence of amplified HER2 has been demonstrated in circulating cfDNA during follow-up.⁷⁷ This led the researchers to propose such amplification as a marker for prognosis and response to treatment with monoclonal antibodies, such as trastuzumab.⁷⁷ A recent study has provided proof of the concept that tumor cfDNA circulating in plasma represents a highly sensitive biomarker of tumor burden in metastatic breast cancer.⁷⁸ Specifically, the researchers developed new methods to identify somatic genomic alterations (point mutations, structural variants) and designed personalized assays to quantify tumor cfDNA circulating in plasma from 30 patients. They found that levels of tumor cfDNA showed a greater dynamic range and greater correlation with changes in tumor burden than did CA15-3 or circulating tumor cells.⁷⁸

Many chemotherapeutic agents act on pathways in which KRAS, BRAF, EGFR, or p53 are involved.^79,80 For this reason, in many cases, it is important to know the mutation status for predicting the response to treatment and monitoring the disease. The analysis of plasma offers a noninvasive and quick way to find out this information and, in this sense, it has been reported that detection of EGFR mutations in plasma might be useful to predict disease progression, disease-free intervals, and drug resistances in patients with lung cancer.^79–81 Moreover, it has recently been reported that sequencing of cancer exomes in serial plasma samples might be useful to track genomic evolution of metastatic cancers in response to therapy.²⁶ The researchers described a noninvasive approach for characterizing cancer exomes in plasma that might enable detailed and comprehensive evaluation of clonal genomic evolution associated with treatment response and resistance.²⁶

Summarizing, patients whose tumors have specific mutations might be monitored by analyzing the tumor cfDNA in their plasma samples.^10,26,78

Gene hypermethylations

Some tumors are related to specific epigenetic alterations (eg, methylation), leading to changes in the expression of promoters of suppressor genes, which results in their silencing. Such alterations occur early in tumorigenesis and in DNA fragments that are rich in cytosine and guanine (CpG islands). The first observations were reported in 1999 by Esteller et al⁸² in non-small cell lung carcinoma; now, the detection of these alterations represents one of the most promising advances in cancer diagnosis.

Epigenetic alterations are not tumor specific; moreover, there are some genes that are frequently hypermethylated and silenced in different types of tumors. In fact, many studies have analyzed panels of genes to increase sensitivity.^83–85 Thus, it is essential to select accurately the genes to analyze for each type of cancer to improve the sensitivity of the analyses. Some of the most common aberrant methylations affect the p16 tumor suppressor gene and have been found in patients with liver, lung, and breast tumors.^82,86,87 Other suppressor genes frequently hypermethylated are SEPT9, RASSF1A, GSTP1, and DAPK, among others.

The clinical value of the detection of hypermethylated genes in plasma has been shown in different types of cancer such as breast,^84,85 colon,⁵³ liver,⁸⁸ esophagus,⁸⁹ and urological tumors,⁹⁰ as well as in hematologic diseases.⁹¹ It has been suggested that the methylated status of cfDNA circulating in plasma might be a tool for prognosis stratification and the prediction of the response to some chemotherapeutics agents,^8,9,92 disease-free interval, and risk of relapse.^53,88,89

Hypermethylations have also been detected in cfDNA from other body fluids such as urine.⁹⁰ Specifically, the presence of tumor cfDNA has been reported in 70% of urine samples, suggesting its utility for the diagnosis and monitoring of patients with urologic tumors such as bladder, prostate and kidney cancers.⁹⁰

Microsatellite alterations

“Microsatellites” are repeating sequences of one to six nucleotides that are scattered along the genome; their function is unknown. Such sequences serve to identify “loss of heterozygosity” (LOH), which is a frequent alteration of tumor DNA characterized by the loss of an allele when compared with matched normal DNA from the same individual. LOH indicates the absence of a functional tumor suppressor gene by deletion. In contrast, some tumors have abnormally long or short microsatellites as a result of a defective DNA repair process; this is termed “microsatellite instability” (MSI).

It has been reported that LOH and MSI can be detected in cfDNA circulating in plasma; however, discrepancies between tumor DNA and plasma cfDNA have been reported.² Although the detection of such alterations in plasma is more probable in advanced stages, some researchers have suggested a potential value for such detection at the diagnosis stage of breast⁹³ and ovarian cancers,⁴⁴ which may have an even higher sensitivity than the quantitation of cfDNA.⁴⁴

To increase the sensitivity of this kind of marker, they have been tested in combination with several other plasma markers, such as methylations and prostate-specific antigen in prostate cancer.⁹⁴ In addition, panels of microsatellites are often used to improve sensitivity.^95,96 For example, in a recent prospective study in breast cancer patients, LOH was determined by PCR-based microsatellite analysis using a panel of eight polymorphic markers.⁹⁶ The researchers found that LOH at those markers was significantly correlated with tumor stage, tumor size, lymph node metastasis, positive progesterone, and HER2 status.⁹⁶ Moreover, LOH at a marker mapping to cyclin D2 correlated with shorter overall survival. Thus, the researchers concluded that the improved detection of LOH on cfDNA provides important information on DNA losses of tumor suppressor genes (TIG1, PTEN, cyclin D2, RB1, and BRCA1) in breast cancer. In particular, loss of the cyclin D2 gene may become an important prognostic marker easily detectable in the peripheral blood.⁹⁶

Prior to that interesting study, it was reported that the assessment of microsatellite status in plasma might be a useful predictive tool for prognosis in breast carcinoma,⁹⁷ to monitor the response to surgical treatment; in oral squamous cell carcinoma;⁹⁵ and to biochemotherapy in metastatic melanoma.⁹⁸

Viral DNA

Viruses are the main etiologic factors of a number of tumors and can be detected in plasma by PCR-based techniques. In fact, the presence of cfDNA of Epstein–Barr virus (EBV) has been demonstrated in nasopharyngeal cancers, Hodgkin’s disease, and Burkitt lymphoma; human papillomavirus in cervical tumors; and hepatitis B virus in hepatocellular tumors, certain lymphomas, and gastric cancers.^{3,10,12,99–102}

The presence of EBV sequences in plasma has been the focus of many studies, perhaps due to the high incidence of nasopharyngeal carcinoma in Asian countries. It has been shown that the detection of EBV in plasma is a powerful diagnostic tool and its quantitation might have prognostic value.¹⁰³ In addition, detection of EBV in plasma has also been associated with response to radiotherapy, disease recurrence, and survival.¹²

mitDNA

Although the study of the genetic and epigenetic alterations of gDNA is the cornerstone of cancer research, the discovery of specific alterations in mitDNA in cancer patients¹⁰⁴ has opened new routes in the search for clinical tools. The detection of aberrant changes in mitDNA is becoming an important tool for the early diagnosis of cancer, which is in part due to the fact that the analysis of mitDNA has some advantages over that of gDNA.¹¹ These advantages can be summarized as follows.

• The mitochondrial genome is shorter and more simply organized than nuclear DNA. These unique properties make the screening of a mitochondrial genome much easier and more cost-effective.

• mitDNA’s high number of copies, in comparison to nuclear DNA, make it a much more sensitive method.

• mitDNA fragments have been detected in different body fluids from cancer patients at early stages, such as in the blood^104,105 saliva,¹⁰⁶ urine,¹⁰⁷ and sputum.

The alteration of mitDNA as a response to adaptation changes was first described in the early twentieth century, when it was termed the “Warburg effect.”¹⁰⁸ Alterations in mitDNA have been found in the plasma of healthy individuals¹⁰⁹ and have also be related to tumor development and progression.¹¹⁰ The alterations, which are point mutations, deletions, insertions, and quantitative changes,¹¹ have been detected in a wide range of tumors, such as breast, colon, liver, head and neck and lung.¹¹¹ It is important to highlight that the identification of mutations in a concrete region typical to many tumors is referred to as the “D-loop,” which may have diagnostic value.¹⁰⁴

The presence of mitDNA mutations has been reported in plasma and serum samples from patients with hepatocellular, pancreatic, prostatic, colorectal, and esophageal cancers, among others.^112–116 Moreover, several studies have suggested that quantitation of mitochondrial cfDNA in plasma might serve as a clinical tool,^117,118 and may even have a higher diagnostic value than gDNA in some cases.¹¹⁸ Specifically, Kohler et al¹¹⁷ compared the levels of gDNA and mitDNA in plasma from patients with benign and malignant breast tumors with those from healthy controls. They concluded that both nuclear and mitochondrial cfDNA have potential as biomarkers in breast tumor management; however, the nuclear cfDNA showed greater sensitivity and specificity. Zachariah et al¹¹⁸ conducted a study in 104 women with ovarian cancer, benign tumors, and endometriosis, and compared the levels of gDNA and mitDNA in plasma in these patients. Patients with epithelial ovarian cancer had significantly higher amounts of nuclear and mitochondrial cfDNA than the other women, but the levels of cfDNA in plasma were related to neither pathological parameters nor CA125 levels. In addition, the researchers found that quantitation of mitDNA was a unique way to differentiate between patients with ovarian cancer and endometriosis.¹¹⁸

Other studies have used “mitochondrial DNA integrity,” which has been defined as the ratio between long and short fragments of mitDNA.¹¹⁹ It has been reported that such an index might differentiate between patients with urologic malignancies (renal, prostate, and bladder tumors) and healthy subjects, with a sensitivity of 84% and a specificity of 97%.¹¹⁹

Finally, it has been reported that some mitDNA polymorphisms are associated with cancer development, thus a genetic analysis of such polymorphisms could help to identify target populations to establish screening programs.¹¹