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Circulating DNA

What We Know and What We Need to Learn

DAVID SIDRANSKYa

The Johns Hopkins University, Baltimore, Maryland 21205, USA

HISTORY

Cancer is driven by the accumulation of genetic changes that parallel the progres-sion of preinvasive to invasive lesions. These genetic changes occur over many yearsand lead to the evolution of extended clonal patches in affected patients. These largepatches provide many opportunities to detect the presence of neoplastic cells in bodi-ly fluids that drain or bathe the affected organs.1 Yet blood, the only fluid in directcontact with all bodily organs, remains the most attractive for cancer detection. Thisknowledge has driven waves of investigation to develop better and more precise can-cer markers that can be directly measured in the blood. Among these cancer markers,none may be more puzzling or promising than circulating DNA derived from the pri-mary tumor. From the articles that follow, it will become apparent that circulatingDNA is easily accessible and amenable to multiple molecular detection approaches.

The concept of circulating free DNA is not new. The possible presence of circu-lating DNA was first revealed more than 50 years ago. It was not until the late 1970sthat precise quantitation of free DNA in the serum of patients with cancer was car-ried out. These studies, predominantly pioneered by Leon and Shapiro, revealedhigher levels of plasma and serum DNA in patients with cancer compared to thosewith benign disease.2,3 They also demonstrated that patients with metastatic diseasetended to have even higher levels, sometimes reaching microgram quantities of DNAper milliliter of plasma. It was not clear at that time if the DNA was derived from theprimary tumor because other diseases such as autoimmune diseases also demonstrat-ed higher levels of serum DNA. Because of this, simply measuring the level of DNAwas never established as a definitive approach for detection or monitoring of cancerpatients. Approximately 10 years ago, Stroun and Anker began to characterize thiscirculating DNA with strand stability assays and demonstrated for the first time thatit was likely to be neoplastic DNA.4 In the mid-1990s, detection of mutated ras se-quences in the serum of patients with solid tumors was definitively demonstrated bySorenson and others.5 This was followed up very quickly by microsatellite analysisof serum in patients with small cell lung cancer and head and neck cancer.6,7 Theselatter studies established that the entire spectrum of genetic changes seen in primarytumors could also be detected in the serum of patients with solid tumors. In the lastfew years, there has been an explosion of new studies demonstrating various geneticchanges in the serum or plasma DNA of patients with primary cancer.

aAddress for correspondence: HNCR/Sidransky Lab, The Johns Hopkins University, 720 Rut-land Avenue, Suite 818, Baltimore, MD 21205. Voice: 410-502-5155; fax: 410-614-1411.

dsidrans@mail.jhmi.edu

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TARGETS

Recent studies have all confirmed that any somatic genetic alteration in a primarytumor is a potential target for molecular detection in the serum or plasma. The mostcommon target in recent studies has been the K-ras oncogene, which is mutated in alarge number of GI tumors and in approximately one-third of adenocarcinomas ofthe lung. It is clear that K-ras mutations cannot be identified in the serum of everypatient with a K-ras mutated tumor. Because single-copy gene mutation detectionapproaches are quite sensitive, it is quite possible that some primary tumors do notrelease DNA into the blood for a variety of mechanical and physiological reasons.Technical reasons for lack of sensitivity as well as the potential for false positivesare described in many of the papers that follow.

Another potential target is LOH (loss of heterozygosity) and repeat sequence in-stability assayed through microsatellite analysis. The high informativity of micro-satellite markers has made them a favorite of somatic geneticists who map tumorsuppressor genes and their use has led to the identification of several minimal areasof loss in primary tumors. Although widespread microsatellite instability is rare intumors that do not have mismatch repair deficiency, selected microsatellites appearto be vulnerable to much rarer microsatellite alterations and have been described inseveral tumor types. As noted above, two recent papers described LOH and micro-satellite instability in the serum and plasma of patients with head and neck cancerand small cell lung cancer. Virtually every tumor type harbors several areas of LOHthat have been carefully mapped, as well as microsatellite instability at a few select-ed markers. Microsatellite alterations have now been described in the serum or plas-ma of patients with head and neck cancer, lung cancer, renal cancer, breast cancer,and other tumor types. However, unlike assays that detect single-copy point muta-tions in circulating DNA, LOH cannot be precisely identified if most of the serumDNA is derived from normal cells, while microsatellite instability has a sensitivityof approximately one in a hundred.

Promoter hypermethylation was described over a decade ago, resulting in the lackof transcription and probable inactivation of the retinoblastoma gene. Further studiesdemonstrated that promoter hypermethylation of the VHL gene played a role in so-matic inactivation of this critical tumor suppressor gene in renal cancer.8 In 1995, itbecame apparent that p16 (CDK inhibitor) mutations were rare, and promoter hyper-methylation resulting in gene inactivation was found to be common in a variety ofdifferent primary tumors.9 Importantly, this inactivation was found to be reversiblewith demethylation treatment, leading to reconstitution of p16 transcription and sup-pression of cell growth. A small revolution has led to the elucidation of new markerssince multiple genes are now known to be somatically inactivated by promoterhypermethylation. Because of a recently developed methylation-specific PCR assay(MSP), sensitive detection of hypermethylated cells can now be carried out in pri-mary tumors and serum. Recent studies have highlighted the use of promoter hyper-methylation in the detection of lung cancer and head and neck cancer.10,11

Undoubtedly, as more methylated targets are found, other studies will follow in dif-ferent tumor types.

Viral oncogenesis predates our current understanding of endogenous genetic al-terations in the progression of human tumors. The initiation of cervical cancer by hu-man papilloma virus (HPV) has been well established, as has Epstein-Barr virus

3SIDRANSKY: CIRCULATING DNA

(EBV) as a major cause of nasopharyngeal cancer. Integration of viral DNA isthought to be critical for the transformation and continued proliferation of some ofthe early precursors to these cancers. It therefore stands to reason that integrated vi-ral DNA will also be present in the serum or plasma of patients with primary tumorscaused by viral infection. Recent studies have documented the presence of EBVDNA in the plasma and serum of patients with nasopharyngeal cancer.12,13 Unpub-lished studies (D. Sidransky) suggest that HPV DNA can be found in at least a per-centage of patients with head and neck cancers where the primary tumor harborsHPV DNA. Recent quantitative studies have shown that EBV DNA may be an in-valuable tool for the monitoring of these patients in search of tumor recurrence.14

FUTURE DIRECTIONS

The initial enthusiasm surrounding the identification of free circulating DNA wasdampened by the lack of available molecular techniques to document its origin. Ithas now been proven that, at least in some patients, circulating free DNA harborsidentical changes to that seen in the primary tumor and therefore reflects the pres-ence of circulating tumor DNA. We do not yet understand the mechanism of how thisDNA is released into the serum or plasma. It is even unclear if this DNA reflects cir-culating cells destroyed by the immune system or whether free DNA is released intothe circulation in a fashion similar to that seen for protein markers. A summary ofthe studies presented here suggests that patients with advanced disease and thosemost likely to have circulating cells are generally positive for any of these DNAmarkers in a variety of different cancers. This fact suggests that circulating tumorcells are likely to contribute to the levels of circulating free DNA. However, it wasalso clear from a variety of studies that patients with very early stage disease can alsoharbor free circulating DNA. In at least one case, a carcinoma in situ with promoterhypermethylation was found to share the identical alteration in the free circulatingDNA.11 These studies raise significant issues about the biology and physiology ofhow the DNA is released and maintained in the circulation and ultimately on its clin-ical value.

There appear to be two major clinical avenues to pursue for the use of circulatingDNA markers. The first and most proven is the use of circulating DNA to monitorfor disease recurrence or to establish disease-free status. As mentioned above, tan-talizing evidence with quantitative EBV DNA analysis suggests that circulatingDNA markers will perform amiably in this regard. Anecdotal reports have alreadysuggested a correlation between the presence of circulating DNA genetic alterationsand clinical follow-up for the absence or presence of tumor recurrence. However, itis abundantly clear that large prospective studies with longitudinal follow-up are es-sential if we are to carefully evaluate these circulating DNA markers and eventuallyintegrate them into the clinical setting.

A long-term goal of all physicians is the development of a simple blood test tofind early stage cancers that are amenable to surgical resection and cure. Geneticallyaltered circulating DNA is a candidate marker for such early detection approachesin high-risk patients or even for screening of larger populations. At this time, how-ever, a significant amount of work remains in selecting the best markers for eachtumor type and the selection of appropriate populations for such detection and

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screening approaches. Circulating DNA markers offer the promise of precise quan-titative analysis without the need to establish difficult cutoffs as is necessary forprotein markers. Ultimately, the further development of high throughput assays willenable the testing of these molecular assays in prospective studies that can betteranswer these questions. The recent flurry of scientific and clinical studies in this areasuggests that circulating DNA will be a favorite research target well into this newmillennium.

REFERENCES

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5. SORENSON, G.D., D.M. PRIBISH, F.H. VALONE, V.A. MEMOLI, D.J. BZIK & S-L. YAO.1994. Soluble normal and mutated DNA sequences from single-copy genes in humanblood. Cancer Epidemiol. Biomarkers Prev. 3: 67–71.

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