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Detection of Mutated KRAS2 Sequences as Tumor Markers in Plasma/Serum of Patients with Gastrointestinal Cancer

Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 03756

ABSTRACT

Mutated KRAS2 commonly can be detected in the plasma/serum of patients with pancreatic or colorectal cancers possessing this mutated gene. Positive assays are more common in patients with higher stage tumors but some smaller cancers can also be detected; occasionally, patients with large tumors have negative assays. Because relatively few patients with low-stage tumors have been evaluated, more studies in patients with smaller tumors are needed to further define the clinical usefulness of these assays. The reasons for variable results, particularly in patients with larger tumors, is unclear, although a variety of factors may be involved. More sensitive assays need to be developed that will increase the detection rates, although the problem of producing false positives must be minimized. The presence of mutated KRAS2 sequences in the plasma/serum seems to be quite specifically associated with the presence of cancer containing this mutated gene. This is an important feature of KRAS2 as a tumor marker. Preliminary studies in patients with pancreatic cancer suggest that assays for mutated KRAS2 can complement the commonly used CA19-9 assay and provide additional clinically useful information. The results from currently completed studies on the detection of mutated KRAS2 in patients with colorectal and pancreatic cancer are promising, and the potential usefulness of KRAS2 as a clinically important tumor marker should encourage future research.

Introduction

The evaluation of free DNA (non-cell associated) in the blood has passed through several phases during the past 50 years. In 1948, Mandel and Métais (1) reported the presence of free DNA and RNA in the plasma of 25 individuals. Whether their methods were reliable is open to discussion, but at least they posed a fundamental innovative question, "Is there non-cell associated DNA (and RNA) circulating in the blood?" Their study introduced the era of qualitative studies on plasma/serum DNA. Subsequently, additional studies confirmed the presence of free DNA both in normal blood (2) and particularly in certain diseases such as systemic lupus erythematosis (3 , 4) and cancer (5, 6, 7, 8) .

There was also a search for specific DNA sequences in the plasma. The first successful report in this area was by Li and Steinman (9) , who described an increase in Alu sequences in comparison with cellular genomic DNA in plasma DNA from patients with systemic lupus erythematosis. This sequence-specific research was taken an important step further in 1992 when Martin et al. (10) reported that gene-specific sequences, i.e., HLA class II gene sequences, could be detected in plasma almost as well as in genomic DNA derived from lymphocytes.

The focus of this review is on studies detecting mutated KRAS2 sequences in plasma/serum from patients with gastrointestinal cancer. Mutated RAS genes were the first tumor-specific gene sequences detected in the blood from patients with cancer (11 , 12) . These early RAS papers described the presence of NRAS in the blood of patients with leukemia and myelodysplasia and the presence of mutated KRAS2 sequences in the blood of patients with pancreatic cancer. They stimulated the current actively pursued search for specific tumor-derived DNA sequences in the plasma or serum and have opened a broad new area of research.

Mutated KRAS2 sequences are particularly attractive tumor markers in blood for several reasons:

(a) They are found frequently in several types of commonly occurring human cancers, such as adenocarcinoma of the pancreas, colorectum, lung, and thyroid. These cancers resulted in 1.5 million deaths, which represented 30% of the cancer deaths in the world in 1990 (13) . Point mutations in KRAS2 genes may also occur less commonly in testicular tumors (14) and in carcinoma of the gallbladder (15) , stomach (16) , endometrium (17) , and ovary (18) , as well as in certain types of hematological malignancies (19 , 20) .

(b) When mutated KRAS2 sequences are detected in blood, they seem to be associated quite specifically with cancer.

(c) Finally, the KRAS2 mutation is usually at one site, i.e., the 12th codon, so that detection assays can be focused on this position and are, therefore, simpler.

Probably as a result of these characteristics, there have been more completed studies of the mutated KRAS2 sequence as a tumor marker in blood than for any other DNA sequence (12 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) .

The basis and background for this review are the above-mentioned series of reports on the detection of mutated KRAS2 sequences in the plasma/serum from patients with colorectal cancer or pancreatic cancer, representing the results of studies from nine different laboratories.

Results and Discussion

A summary of the results of assays for detecting mutated KRAS2 sequences in plasma/serum from patients with colorectal cancer in these studies include the evaluation of 131 patients (Table 1) . In 31 patients (24%) of this group, the results of the assays were positive. This is a lower incidence than might be anticipated ideally because 50% of all patients with colorectal cancer would be expected to have primary tumors containing mutated KRAS2 genes (32 , 33) . The patients who were positive tended to have more extensive tumors, and most had metastatic cancer.

(a) The results appear to be similar in both series of patients, i.e., patients with colorectal and with pancreatic cancer, because in both groups, it appears that some patients who might be expected to have tumors containing mutated KRAS2 genes have negative plasma assays for the mutated sequence.

(b) The positive assays reported in these studies tend to be in patients with higher stage disease. But, at least to some extent, this pattern is dependent upon the type of case that is being evaluated and, in some series, all of the patients included in the studies had higher stages of disease. Therefore, in those series, the positive cases necessarily would have higher stage tumors.

The most unambiguous data are contained in the summary of results from the eight different laboratories that have evaluated the detection of the presence of mutated KRAS2 in plasma/serum from patients who have colorectal and pancreatic cancers containing mutated KRAS2 (Tables 2 and 4) . In these data, the denominator is certain; all these patients have tumors containing mutated KRAS2 genes. The numbers are relatively small, but the results are quite consistent among these different studies. The summary tables indicate that there are positive assays only in 50% of both types of patients, although the groups are composed largely of patients with more advanced cancers.

Considering this, it is more meaningful to look at the summaries of data from the patients who had positive tumors but who had negative plasma assays (Tables 5 and 6) . Although the size of the groups represented in these summaries are relatively small, two observations can be made: (a) some of the negative assays were in patients with advanced tumors, both in the series of patients with colorectal cancer as well as in the series of patients with pancreatic cancer; and (b) most of the negative results occur in patients with lower stage tumors, although only a few patients with small tumors were evaluated. Clearly, more data from patients with lower stage disease are needed to arrive at any definite conclusions concerning the usefulness of KRAS2 assays of plasma/serum in patients with small tumors.

Is the non-cell associated DNA in the plasma/serum derived directly from the tumor or is it derived from the breakdown of circulating metastatic cells? This is a controversial issue but one well worth considering, because the derivation of the tumor DNA can influence the whole approach to developing assays for detecting mutated sequences in blood. It is well established that in patients with malignant tumors, metastatic tumor cells circulate in the blood at some stage in the life of the tumor. Yet, they do not always result in the development of metastatic tumors. Nonetheless, the available evidence indicates that the circulating free DNA is more likely to come directly from the tumor. In an appealing model, the DNA derived from apoptosis of neoplastic cell leaks directly into newly formed permeable lymph and/or blood vessel systems of the tumor and, hence, into the circulation.

Part of the evidence supporting this model is the size of the DNA fragments in blood. If the circulating DNA is coming primarily from metastatic cells in an ongoing way, one would expect to find DNA fragments with a variable spectrum of sizes, including large fragments derived from these cells as they are gradually broken down. However, the evidence from both gel electrophoresis and electron microscopy studies shows that practically all of the plasma DNA is composed of relatively small fragments of <1500 bp (43) . On the other hand, it is conceivable that the DNA from the circulating metastatic cells could be broken down by apoptosis intracellularly and then released as small fragments that are found ultimately in the plasma. The latter possibility is unlikely, because the number of circulating metastatic cells seems to be inadequate to explain the observed amount of tumor DNA in the plasma/serum. Even if the tumor-derived DNA concentration is only 5 ng/ml, this represents the DNA from 850 tumor cells. Such a concentration of circulating metastatic tumor cells is unlikely (44) .

Perhaps an even more compelling reason for suspecting that plasma DNA comes directly from apoptosis in the tumor is the large amount of circulating free DNA that can be detected in the plasma when, in these same patients, circulating metastatic cells are not found. Aihara et al. (45) used reverse transcription-PCR and a keratin marker for epithelial cells. This assay was positive in 12 of 12 (100%) pancreatic cancers. However, they rarely found positive cells in the peripheral blood from 40 patients with pancreatic cancer, although 75% of whom had stage 3 or 4 tumors. One would have expected that these same patients would have significant amounts of tumor DNA in their circulation. Castells et al. (29) also were unable to detect circulating tumor cells in 28 patients with pancreatic cancer that had demonstrable circulating mutated KRAS gene sequences derived from their tumors. Thus, the weight of available evidence is opposed to the possibility that plasma DNA is derived from circulating metastatic tumor cells.

The previously described summary of studies (Tables 1 2 3 4 5 6) indicates the current status of research efforts, up to the recent past. It shows that until now, assays to detect mutated KRAS2 in plasma are negative in many patients who have tumors containing this mutated gene. Considering this, one can view this as representing a glass that is half full or a glass that is half empty. Mutated KRAS2 can be detected in the plasma/serum from 50% of patients with positive tumors, but mutated KRAS2 sequences were not detected in the plasma/serum of the remaining 50% of patients, even when most of them had rather advanced tumors. The results are surprising and disappointing, because the early, small studies suggested that mutated KRAS2 could be detected in a greater percentage of these patients than has actually occurred to date. By widening the search to include other tumor- derived sequences, perhaps we can greatly increase this detection rate.

Despite these results, mutated KRAS2 has the potential to be one of our most useful tumor markers because of its cancer specificity in plasma/serum. For this reason alone, it is desirable to consider the factors that may influence assays for mutated KRAS2 in the hope that a broader discussion will lead us to methods that will ultimately improve the sensitivity and specificity of our assays. These considerations are derived primarily from our experience over the past 7 years with the ASA² assay for the detection of mutated KRAS sequences in plasma. This is an assay that was simultaneously described in reports from five different laboratories in 1989 (46, 47, 48, 49, 50) .

The above-mentioned summary of recent reports on pancreatic cancer indicates that tumor-derived mutated KRAS2 sequences are detectable mainly in patients with relatively advanced tumor burdens. Thus, these conclusions suggest that detecting mutated DNA sequences in plasma is not a very sensitive assay for determining the presence of early cancer. However, there have been some exceptions, as indicated previously (25 , 27 , 31) .

Unfortunately, the current reports indicate that mutated KRAS2 is not found in plasma from many patients, although they have cancers containing mutated KRAS2 sequences. Why does this occur? Is it because mutated KRAS sequences are not there, or are they present and not detected? If they are not present in a detectable amount, are they not released into the blood? Are they released and rapidly cleared? Or, on the other hand, are the mutated sequences present but not detected in some patients because of the limited sensitivity of our current assays?

Factors That May Affect the Release of Tumor DNA into Plasma.
The first possible answer is that there may be few of the mutated KRAS2 sequences in the circulating plasma in some patients with tumors containing mutated RAS genes. How can this be rationalized? Is it because they are not released, or is it because they released and too rapidly cleared? Of course, there may be fewer tumor-specific sequences in the plasma/serum because the tumor is small. Presumably, very small tumors will release only a small amount of tumor DNA into the plasma, whereas large tumors will yield a larger amount. Additionally, the amount of tumor DNA may vary, depending upon the biological characteristics of the tumor. There are known differences in growth rates and histological grade (i.e., malignancy) that, along with differences in rates of apoptosis and necrosis, may lead to more or less tumor DNA in the blood, even with tumors of the same size. Vascularity, as well as local growth factor influences, may also affect the release of tumor DNA into the circulation (51) . Moreover, there may be other biological differences among tumors that have yet to be identified and that may affect the release of tumor DNA into the blood.

Catabolism of Plasma/Serum DNA.
A second possibility is that DNA is released by the tumor and that there may be variations in the clearance of DNA from the blood, which may lead to the presence of more or less DNA remaining in the blood. Relatively little is known about catabolism of extracellular DNA in the blood. In 1989, it was reported that, after hemodialysis in patients, the half-life of DNA in the blood was 4 min (52) . In more recent studies of pregnant women, it was determined that the mean half-life of circulating fetal DNA was 16.3 min (53) . Plasma nucleases do not seem to be involved primarily in catabolism. A detailed study by Eder et al. (54) in 1991, using plasma from several animals as well as from humans, indicates a lack of nuclease activity in the plasma that might have a significant effect on double-stranded DNA of the size found there. More recent studies also indicate that plasma nucleases do not play a major role in the catabolism of extracellular DNA in the blood (53) .

Could it be that extracellular DNA in the blood is excreted by the kidney? There is little definitive information about this. Increased urinary DNA was described in patients with acute pancreatitis as early as 1967 (55) . Electron microscopic studies of urinary DNA from three normal men indicate that the size of the DNA fragments overlaps the size range of the shortest fragments of the plasma DNA.³ Thus, the size of the urinary DNA is consistent with the possibility of renal excretion. However, we were unable to detect specific tumor-derived gene sequences in the urine from patients with pancreatic cancer who have mutated KRAS2 sequences in their plasma.⁴ More recently it has been reported by Y. M. D. Lo⁵ that he was unable to detect Y chromosome sequences in the urine of women carrying male infants, although the Y chromosome sequences were present in the mother’s plasma. Therefore, definitive evidence for renal excretion of plasma DNA is lacking at this time.

Factors That May Affect the Sensitivity of Assays for KRAS2.
The other possibility for the apparent lack of sensitivity in detecting mutated sequences is that although tumor DNA is commonly present in the serum/plasma of patients with cancer, our current assays are not able to detect it. A variety of assays were used in the studies by the nine laboratories that have been reviewed (Table 9) . Mutated KRAS2 sequences were detected in both plasma and serum in these studies. A priori, plasma would seem to be the better starting material because DNA may be released from WBCs into the serum during the clotting process. This increase in background WT DNA in serum may interfere with the results of some assays for mutated KRAS2 sequences.

A particularly relevant observation regarding this question is the report on seven patients with stage B colorectal tumors, which contained mutated KRAS2 genes (26) . Mutated KRAS2 sequences were not detected in the plasma in any of these seven patients. Nonetheless, investigators were able to detect mutated p53 sequences in five of these same seven patients. It is likely that the tumor DNA was circulating in the plasma of these patients, but the assay for KRAS2 was not sensitive enough to detect it. Thus, depending on which method of assay is being used, it may not be possible to readily detect the tumor-derived KRAS2 sequences present in the plasma.

To evaluate the sensitivity of an assay, one can use model systems of mixtures containing mutated and WT DNA. However, to obtain significant results, one needs to simulate closely the situation in the blood, not only in terms of relative amounts of mutated and WT DNA but also in terms of absolute amounts. An assay may be sensitive enough to detect 1 part mutated sequences in 1000 parts WT if the mixture consists of 10 ng in 10 µg but not be able to detect 10 pg mixed in 10 ng. Each mixture is 1 part per 1000. To be relevant to the detection of mutated sequences in plasma, one must evaluate pg in ng relationships. Furthermore, the reconstructive experiments must be made in plasma/serum to simulate the inhibiting conditions that exist naturally in the blood.

What are the conditions that limit our assay sensitivities? One factor that may limit sensitivity greatly is the amount of background WT DNA. If one is evaluating the detection of mutated genomic DNA alone in a progressive series of dilutions with the ASA assay, one can detect 10 pg. On the other hand, if one mixes mutated DNA with WT DNA to simulate closely what occurs in plasma, the detection level in 10 ng of WT DNA is about 100 pg, i.e., 1 in 1000. However, this is one-tenth as sensitive as when the mutated DNA sequences are assayed alone. For this reason, it was determined that it was important to remove most of the WT DNA prior to using the ASA assay.⁶

The small size of the DNA fragments in plasma also decreases, at least to some extent, our ability to detect mutated sequences by PCR. Electron microscopy indicates that practically all fragments of plasma DNA are less than 1500 bp in length (43) . DNA fragments may be terminated inside the normal span of our PCR primers. The extent of this effect is dependent upon the distance between the primers during the initial amplification by PCR.

Another situation that may affect our ability to detect small amounts of mutated KRAS2 sequences is the described PCR bias toward WT. Barnard et al. (57) demonstrated a 6-fold bias for WT K-ras (and 15-fold for WT p53) when the WT and mutant sequences were amplified separately or mixed in equal proportions before PCR.

Specificity of Assays.
The question of assay specificity is related to at least two major issues: (a) the specificity of detecting mutated sequences in plasma; and (b) the specificity of mutated KRAS2 in plasma when used as a marker for cancer.

Consider the specificity of detecting mutated KRAS2 sequences. False positives can occur with practically all assays. Investigators have usually controlled for this by performing multiple assays on the same specimen and/or by concurrently evaluating control samples. It is likely that the latter approach is less valid, especially considering how variable PCR can be, depending upon variations in the target sequence. For example, in the case of the ASA assay, one can get false positives if the quantity of target DNA is very small, or if there is an abundance of WT DNA present. However, this latter problem is avoided if one eliminates WT DNA as a preliminary step in the assay.

False positives may also occur with the PCR-based mutation enriched RFLP analysis described by Jiang et al. (58) , Levi et al. (59) , and Terhune et al. (60) . With the Jiang-Levy method, mutations can be generated in the first PCR reaction that subsequently will be enriched in the second PCR. Both because of the power of the selection and the second enrichment step, a very few mutated copies generated by mistakes in the first PCR will produce a result that can be interpreted to indicate that mutated sequences are present in the original specimen. The greater the number of cycles in the first PCR, the greater the likelihood of generating mutated KRAS2 sequences, which can be enriched in the second PCR. In the study by Terhune et al. (60) , when >20 cycles were used in the first PCR, there was a significant chance of mutations becoming generated that then would be enriched in the second PCR. It was also found that, by decreasing the concentration of nucleotides and other modifications to the assay, one could decrease the incidence of false-positive results.

The other basis for a false-positive result, in relation to the presence of cancer, is not related to the type of assay used but, rather, to the possibility that there may be circulating mutated RAS sequences in persons who do not have cancer. This possibility must be considered carefully, because mutated KRAS2 sequences have been demonstrated convincingly in noninvasive proliferative lesions in both the pancreas and colon.

In the pancreas, mutated KRAS2 sequences have been demonstrated in hyperplastic ductal epithelium (35 , 60 , 61) . Does mutated DNA from these ductal epithelial cells get into the plasma? This question is still unanswered, and probably it will be difficult to ever establish this definitely in patients. Nonetheless, a priori, it is doubtful that much, if any, of the DNA from these cells gets into the blood. For one reason, the ductal epithelial cells are surrounded by an intact basement membrane and other periductal connective tissues that tend to separate them from blood vessels. Another even more compelling reason is that cells sloughing from these foci would be expected to readily pass in a more or less intact form into the pancreatic ducts on their way into the intestine.

KRAS2 mutations also have been described in the pancreatic epithelial cells in patients with chronic pancreatitis (62 , 63) . In this case, one would expect the situation to be similar to that in hyperplastic pancreatic ducts. Although in chronic pancreatitis, foci of epithelial cells can get pinched off and separated from the ducts by reactive fibrosis, a priori, it is unlikely that significant amounts of DNA from these cells, which are surrounded by intact basement membranes and dense fibrosis, will get into the blood. Certainly the proliferation and turnover rates of these cells in chronic pancreatitis is significantly less than in pancreatic cancer. Importantly, these expectations are supported by the available evidence. Data from plasma assays for mutated KRAS2 were reported in three studies (27 , 28 , 29) , and mutated KRAS sequences were detected in only 2 of 50 patients with chronic pancreatitis (Table 8) . Nonetheless, the reason for the two positive results in the large series reported by Castells et al. (29) is unclear.

The situation in the colon is probably similar. Mutated KRAS sequences have been demonstrated in a variety of hyperplastic lesions, such as aberrant crypt foci and hyperplastic as well as dysplastic polyps, although only the latter are precancerous (40, 41, 42) . However, it seems unlikely that many of the mutated DNA sequences would get into the blood. Normally, the colonic epithelium is renewed every few days and is separated by an intact basement membrane and the underlying connective tissue from blood vessels. What would be the mechanism for transferring nuclear DNA from superficial epithelial cells into the relatively distant lymph or blood vessels in the submucosa of the colon? What would be the biological role of degenerating epithelial cell DNA passing back into the peripheral blood circulation? Answers to these questions are not readily apparent.

A priori, it is unlikely that tumor DNA passes into the blood circulation until neoangiogenesis occurs in association with invasion of the tumor cells into the submucosa of the colon. On the other hand, cells sloughing from the surface of the normal colon, as well as from superficial hyperplastic and dysplastic lesions, would be expected to readily pass into the lumen of the colon, and subsequently, the cellular DNA would be excreted in the stool. Published reports reviewed here and discussed previously support these latter expectations. In patients with KRAS2-negative colorectal cancers, one would expect that most would have additional polyps as well as other hyperplastic and/or dysplastic lesions in their colons, in addition to their cancers, and many of these abnormal colonic lesions would be expected to contain KRAS2 mutations of variable types (40, 41, 42) . However, a positive assay for KRAS was reported in only 10% of these patients (Table 8) . The explanation for these positive results is unclear. Nonetheless, it seems that if mutated KRAS2 sequences do get into the blood from noninvasive lesions in the colon, the incidence is low.

Thus, summarizing these considerations of sensitivity and specificity, it is desirable in our research efforts to focus on significantly increasing the sensitivity of the assays for mutated KRAS2, while, at the same time, being concerned about false positives. Because there is always a delicate balance between sensitivity and specificity, new types of assays may be needed to achieve this goal. Moreover, it is likely that the matter of specificity of mutated KRAS2, in relation to the presence of cancer, is much less of a problem than sensitivity. When the assays can definitely detect mutated KRAS sequences in plasma, the reviewed reports from the aforementioned nine laboratories suggest that it is probable, or even almost certain, that the patient has cancer.

FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Department of Pathology, Dartmouth-Hitchcock Medical Center, 1 Medical Center Drive, Lebanon, NH 03756. Phone: (603) 650-7644; Fax: (603) 650-6120.

² The abbreviations used are: ASA, allele-specific amplificative; WT, wild type.

³ A. Borges, G. C. Ruben, T. B. Roos, D. H. Porter, and G. D. Sorenson, unpublished observations.

⁴ G. D. Sorenson, unpublished observations.

⁵ Y. M. D. Lo, personal communication.

⁶ G. D. Sorenson, unpublished observations.

Received 12/27/99; revised 3/ 6/00; accepted 3/ 6/00.

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