Chapter 1 Progress and Future Prospects of Modified Nucleosides as Biological Markers of Cancer

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CHAPTER 1 PROGRESS AND FUTURE PROSPECTS OF MODIFIED NUCLEOSIDES AS BIOLOGICAL MARKERS OF CANCER ROBERT w. ZUMWALT~, T. PHILLIP WAALKES~, KENNETH c. K U O ~ ,AND CHARLES W. GEHRKE?

* .4Departmcnt of Biochemistry, University Center, Columbia, MO U.S.A. 65201

of Missouri, and Cancer Rcscarch

*Johns Hopkins University School of Medicine, Baltimore, MD 3Analytical Biochemistry Laboratorics, Inc., Columbia, MO

U.S.A.

U.S.A.

21205

65201

TABLE OF CONTENTS 1.1 Introduction . 1.2 Progress in Modified Nucleoside Studies . 1.3 Prospects: Biomarkers for Leukemias, Lymphomas, and Other Cancers . 1.4 S u m m a r y . 1.5 References.

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1.1 INTRODUCTION The search for biological markers of cancer has mainly focused on three structural categories: hormones, proteins, and nucleic acid components. This chapter will deal with the topic of nucleic acid components as potential biological markers; specifically modified nucleosides. The Introduction to this volume traces the development of reversed-phase HPLC and phenylboronate gel affinity chromatography for the analysis of modified nucleosides in physiological fluids, and the following chapters i n this volume provide detailed accounts of the methods and their applications to various aspects of cancer and normal metabolism research. The term "tumor marker" as coined by Dr. Morton K. Schwartz of the Sloan Kettering Institute refers to some unique metabolic product or

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unusual component of malignant cells which can be measured in body fluids. As emphasized by Borek (ref. 1) and others, the criteria of an effective tumor marker are numerous; it should be specific for malignancy; it should provide a minimum of false-positives and falsenegatives; it should indicate the extensiveness of the malignancy and it should preferably diminish or hopefully disappear after effective therapy. There have been indications for more than 30 years that cancer patients excrete elevated levels of methylated purines and pyrimidines as well as other modified bases and nucleosides (refs. 2, 3). The origin of these compounds was obscure until the discovery of the modification of transfer RNA (ref. 4). The events responsible for the increased excretion of modified nucleosides by cancer patients remain unclear, with increased tRNA turnover, cell death and increased turnover of RNA in the host tissue proposed by various investigators. Investigators have also attempted to elucidate cancer markers which could be utilized to predict which patients are more likely to respond to treatment and which patients have a worse prognosis. Identification of patients with a worse prognosis would perhaps permit the utilization of either more aggressive or innovative forms of treatment for individuals who would not be likely to have a good response to standard approaches (ref. 5). As G. Prodi has pointed out (ref. 6 ) , "a clinical symptom, in this case a tumor marker, is a "sign" that is meaningful only within a previously established theory" (such as for the concept proposed by Borek on altered tRNA metabolism and turnover). Prodi went on to state that "in the field we are now considering, a marker would be an unambiguous "sign" of cancer in the framework of a theory that was previously defined: i.e. a) the specificity of the tumor condition with respect to any other condition in, or occurrence of the b) the link between the considered marker and t h a t organism, and specificity. Only in this way would the data observed "stand for" the tumor - and thus assume the character of the "sign". Unfortunately, such a theory does not yet exist. Cancer research may be defined as an uninterrupted search to define q u a l i t a t i v e l y distinct and specific traits of a tumor cell. What has been defined as

C17 "qualitative" (from Warburg up to the recent immunological studies, and the still more recent oncogene theory) has always been interpreted as "quantitative", that is, as "more or less". The tumor cell is highly mimetic, and it is relatively invulnerable for the same reason as it has such ambiguous markers. Therefore, the history of the study of cancer is also the history of failed markers, or at least markers first held to be absolute, and then relegated to "signs" endowed with a certain ambiguity because they relate to conditions other than cancer. As when we see a cloud on the horizon and do not know whether it is smoke and means fire, or dust and means wind" (ref. 6 ) . At this time, it is generally agreed that presently available biomarkers are ineffective for the primary diagnosis of cancer (ref. 7). Therefore, most recent research on human tumor markers has focused on the surveillance of patients in the post-primary phase of treatment and on comparison of diagnosed cancer patients with non-cancer patients in terms of serum or urine levels of potential biomarkers. This chapter will point to progress and discuss future prospects of modified nucleosides as biological markers of cancer. The Introduction to this volume by Waalkes and Gehrke describes research developments since our involvement in this research, and the other chapters in this volume describe the investigations and provides thorough description, discussions, and reviews of modified nucleosides as biological markers for cancer and normal metabolism. Provided that altered tRNA metabolism is a fundamental aspect of neoplasia, then modified nucleosides resulting from that altered metabolism may indeed be reflective of the neoplastic state.

1.2 PROGRESS IN MODIFIED NUCLEOSIDE STUDIES High performance liquid chromatography with diode array detection (HPLC-UV) has emerged as one of the most popular and powerful techniques for studying the constitutents of nucleic acids, especially in complex samples such as physiological fluids and cell extracts. This chapter will not describe the methodological developments that now permit the accurate measurement of a wide array of major and modified nucleosides in a broad range of sample types; those

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developments are precisely described in other chapters of these volumes, e.g., in Chapter 1, Part A, and Chapter 2, Part C, Gehrke and Kuo describe ribonucleoside analysis by high performance RPLC, and the following chapters in this volume and the references therein provide a thorough description of methodologies for ribonucleoside analysis. Many investigations have been conducted with the general goal of identifying a component of a physiological fluid which would serve as a tumor marker. Concepts, reports, a n d findings which launched investigations of the modified nucleosides as biological markers of cancer have been described in the Introduction and elsewhere in this volume. To add some perspective to the progress of research, a description of the origin and scientific status of the topic of modified nucleosides as biological markers of cancer could be useful. In the period from 1966 to 1972, there were reports that the activities of tRNA-methylating enzymes were elevated in neoplastic cells, that column-chromatographic profiles of tRNAs in neoplastic cells w e r e altered in comparison to their normal counterparts, and preliminary experiments seemed to indicate that bulk tRNA in tumors might be hypermethylated. This information combined with earlier observations that showed elevated excretion of modified nucleobases by cancer patients pointed to the methylated or otherwise modified catabolic products of tRNA as potentially universal biological markers for cancer. W e first published clinical studies of modified nucleosides as potential biological markers of cancer in 1975, using gas-liquid chromatography to measure a very limited n u m b e r of urinary nucleosides (ref. 8-10). That same year, Suits and Gehrke (ref. 11) reported on RPLC analysis of nucleobases and nucleosides, and Gehrke and coworkers followed in 1978 (ref. 12) with the development of the analytical concept which would find widespread acceptance and use in laboratory and clinical research: isolation of nucleosides from complex matrices by phenylboronate gel affinity chromatography followed by their RPLC separation and detection and quantitation by UV absorption. In 1976 Hartwick and Brown had reported on the evaluation of microparticle chemically-bonded reversed-phase column packings for the analysis of nucleosides and their bases (ref. 13). In 1980, Gehrke e t

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al. (ref. 14) further extended the HPLC approach for nucleoside analysis by describing the effects of numerous chromatographic parameters on the separation and quantitation of a number of modified nucleosides. The early studies by Gehrke, Waalkes et al. in the mid-1970s presaged the many research basic and clinical research investigations which would adopt the approach of Gehrke's group for studying modified nucleosides. Numerous research groups in the U.S., Europe, and Japan have studied modified nucleosides and their potential relationships to cancer, and many of these investigations are described or referenced in the chapters of these three volumes. The group headed by F. Salvatore and F. Cimino at the University of Naples has conducted a wide range of studies concerning modified nucleosides and cancer, with much of their work focusing on pseudouridine (ref. 15). Their research on urinary and serum nucleosides has paralleled ours in some ways, and we have obtained very similar results. In addition, we have engaged in collaborative studies with the Naples group, including studies of modified nucleosides in cell cultures and animal model studies (see Chapters 7 and 8, Part C). As described and referenced in the other chapters of this volume, there followed numerous studies on urinary modified nucleoside levels in patients with various cancer types, normal individuals, and patients with diseases other than cancer. Early results from various investigators were mixed, however i t soon became clear that t h e modified nucleosides would not s e r v e as unequivocal universal indicators for the presence or course of all neoplastic disease, but perhaps would function best for specific neoplasias i n following the clinical management of the patient. However, many of the investigations reported i n the literature were encouraging, and in the early and mid-1970s the continuing development of HPLC (pumps, reversed-phase columns, detectors, etc.) offered a much improved analytical approach. The development of a highly specific affinity chromatography method promised to provide a much improved method for isolation of ribonucleosides from the complex matrix of physiological fluids. Since then, progress in

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improving analytical methodologies for accurately measuring modified nucleosides in physiological fluids has been most impressive. The advances in analytical and chromatographic methodology to accurately measure modified nucleosides in physiological fluids has provided investigators with additional research tools to further advance nucleic acid research far beyond the area of biomarkers research (ref. 37). HPLC nucleoside analysis has been developed and extended by Gehrke et aZ. to the quantitative analysis of the complete tRNA molecule, with special emphasis on the extensive and complex modifications present in tRNA (see Chapter 1, Part A), improved and interfaced characterization methods (HPLC-UV, MS, NMR, and FT-IR) for elucidating the structures of unknown modified nucleosides (see Chapter 5 , Part A), developed methods for quantitatively studying the cap structures of messenger RNA (see Chapter 8, Part A), and to a rapid and accurate methodology for studying methylation of DNA (Chapter 10, Part B). 1.3 PROSPECTS: BIOMARKERS FOR LEUKEMIAS, LYMPHOMAS AND OTHER CANCERS. In 1983, Heldman et al. (ref. 16) reported a study of the urinary excretion of modified nucleosides by patients with chronic myelogenous leukemia (CML). They measured urinary modified nucleosides in 15 patients with Philadelphia chromosome-positive CML and determined the correlation with activity of CML. They found that patients in the stable phase of CML had excretion levels one to two times normal, whereas patients in the blastic phase showed elevations up to 12 times normal. The modified nucleosides showing the most significant differences i n excretion between the stable phase and blastic phase were 1-methylinosine, pseudouridine, and N2, N2-dimethylguanosine. Serial nucleoside determinations were made in two patients with CML and found to correlate closely with disease activity. They noted that the degree of elevation and the correlation with disease activity suggest the potential value of quantitation of urinary nucleosides in monitoring patients with CML; in particular, nucleoside excretion may be useful in detecting early blastic transformation.

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Also in 1983, Rasmuson et al. (ref. 17) evaluated urinary pseudouridine as a biologic marker for patients with bronchogenic carcinoma, and reported elevated levels that paralleled clinical stage. Oerlemans and Lange (ref. 18) reported a study of major and modified nucleosides excreted by patients with ovarian cancer. The patients were divided into three groups: benign, borderline, and malignant. They reported that 44% of the measured marker levels of the benign group were in the normal range, whereas 97% of the borderline and malignant groups were outside the normal range. Nielsen and Killman (ref. 19) studied the excretion of pseudouridine and p -aminoisobutyric acid by patients with acute and chronic myeloid leukemia, and compared those levels to healthy control subjects. Pseudouridine excretion was elevated i n over 80% of the patients with untreated AML and CML, and the levels decreased following treatment. Rasmuson and Bjork (ref. 20) studied pseudouridine excretion by 48 patients with malignant lymphomas, and found elevated levels in 50% of patients with histiocytic lymphoma, 33% of patients with lymphocytic lymphoma and 13% with Hodgkin's lymphoma. However, no correlation could be made between level of excretion and clinical stage, and no prognostic value could be attributed to initial excretion levels of pseudouridine. In a later report (ref. 21) Rasmuson and Bjork studied 39 patients with non-Hodgkin's lymphoma before treatment, and found 57% of the patients with highly malignant lymphomas had elevated pseudouridine compared to 28% of patients with low-grade malignancy and 4% for healthy adults. In 1984, Mackenzie et al. (ref. 22) reported their study of RNA catabolites as cancer markers. They found that rats with aflatoxininduced nephroblastomas excreted elevated amounts of urinary modified nucleosides and bases which are catabolites of tRNA. Their study of nucleoside excretion profiles suggested the possiblity for distinguishing between tumors, and their findings indicated that the source of the elevated nucleoside levels may be the host's tissue RNA. Their preliminary studies on humans with lung cancer showed marked elevation of one or more urinary RNA catabolites, and they suggested

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that measurement of urinary RNA catabolites may be useful in the diagnosis, prognosis, and evaluation of therapy in patients with lung cancer. Esposito et al. (ref. 23) evaluated the relationship between increased pseudouridine excretion and retroviral cell transformation. They studied the effect of retrovirus infection and/or tranformation on the rate of pseudouridine excretion by chick embryo fibroblasts. Their results showed that: (a) pseudouridine excretion by chick embryo fibroblasts transformed by Rous sarcoma virus is several times higher than that of normal cells; (b) this increased excretion precedes the appearance of morphological signs of transformation and i t is always present when neosynthesized infectious viral particles are released into the culture medium; and (c) pseudouridine excretion was also increased in cells infected by a mutant of Rous sarcoma virus (RAV-1) which, lacking the src gene, does not transform the cells but replicates normally. In research with Dr. T. Heyman of the Institut Curie (ref. 24), we analyzed modified nucleosides in tRNAs from chicken e m b r y o fibroblasts (CEF), normal and infected with either a wild strain of Rous sarcoma virus, SR-RSV subgroup A (SRA), or a temperature-sensitive transformation mutant (tsNY68). An increased modification in tRNA from SRA-infected CEF cells over normal CEF cells was observed at both the exponential and stationary growth phase. In contrast, n o significant differences were observed in normal CEF tRNA modification levels in relation to the growth phase. We also found that there was a higher increase of modification in tRNA from SRA-infected cells in the stationary phase as compared to that of the exponential phase. Such a difference would be related to the degree of transformation. The tsT mutant (tsNY68) normally replicates but fails to transform cells at high temperature, 42°C. N o increase in the levels of modification was observed in tRNA from tsNY68-infected CEF as compared to tRNA from normal cells, both grown at 42°C. The increase in all detected tRNA modifications (except Q and Y) in transformed cells in comparison to normal cells thus seems to depend on the expression of the src gene (ref. 24).

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Studies of the origin of increased modified nucleoside levels in physiological fluids, such as those of Esposito et al. a n d H e y m a n mentioned above, illustrate the difficulties researchers have encountered in ascribing a source to the nucleoside elevations of patients with cancer. There is still no metabolic mechanism which has been clearly identified as responsible for observed elevated excretion of modified nucleosides by patients with neoplasias. Specifically, in research with Dr. Heyman, we found that the mole percent values of purine and pyrimidine methylated nucleosides in the tRNAs from RSV-infected and transformed CEF cells are 50 to 120% higher than in the tRNAs from non-transformed CEF cells. In addition, the amount of 2'-O-methylated nucleosides and threoninocarbonylated modified adenosine (&A) are only elevated about 10% in RSV-CEF cells over the C E F cells, and the mole percent values of the four major nucleosides found in C E F cells and RSV-CEF cells are essentially identical. This indicates that the only difference between tRNAs from CEF cells and RSV-CEF cells are probably nucleoside modifications. Rasmuson and Bjork (ref. 25) measured pseudouridine excretion in 222 patients with malignant diseases. They found that patients with malignant lymphomas had a 50% frequency of elevated pseudouridine excretion, colorectal carcinomas 25%, bronchogenic carcinomas 3 1%, and in cases of mammary carcinomas 30%. They also reported that excretion increases paralleled increasing clinical stages of the disease. They noted that for patients with bronchogenic carcinoma and possibly malignant lymphomas, elevated pseudouridine excretion is correlated to shorter survival. They concluded that pseudouridine i s a marker of malignancy, and as such it could be used as a complement to clinical stage and to predict the prognosis. Heldman et al. (ref. 26) also studied the differential excretion of modified nucleosides by patients with adult acute leukemia, and reported that the urinary excretion of 1-methylinosine and N2, N2dimethylguanosine may prove to be valuable clinically in following disease activity in patients with acute lymphoblastic leukemia (ALL), and in distinguishing patients with A L L from those with acute myelogenous leukemia (AML).

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Heldman et al. (ref. 27) also studied the relationship of urinary excretion of modified nucleosides to disease status in childhood acute lymphoblastic leukemia (ALL), and demonstrated that excretion of modified nucleosides reflects disease activity in childhood and that the urinary nucleosides may be useful clinical markers for this disease. In 1986, Tamura et al. (ref. 28) studied the urinary excretion of pseudouridine in patients with hepatocellular carcinoma. They reported that the urinary concentration of pseudouridine in the carcinoma patients was significantly higher than in patients with liver cirrhosis or healthy controls. Seventy percent of the 23 patients with hepatocellular carcinoma had urinary pseudouridine levels higher than the mean value for the healthy controls plus 2 standard deviations. When urinary pseudouridine was used in combination with serum alpha-fetoprotein, 83% of the carcinoma patients were positive for marker elevations. Tamura et al. considered urinary pseudouridine and serum alphafetoprotein to serve as complementary markers for diagnosis of hepatocellular carcinoma. Tamura e l al. (ref. 29) also evaluated urinary pseudouridine as a tumor marker in patients with small cell lung cancer. They reported that urinary pseudouridine is not a specific marker for SCLC, but it relates to the tumor burden and reflects the clinical status of patients. They found that in the limited number of cases examined, the positivity rate for urinary pseudouridine concentration was higher than that for CEA, and that when the two markers were combined, the positivity rate is further elevated above that of either single marker. In a study of colorectal cancer patients, Nakano et al. (ref. 30) reported there were no significant differences in the concentrations of pseudouridine, 1 -methylguanosine, N2-methylguanosine, and Nz,N2dimethylguanosine between urine samples taken before and after surgery from eight patients, and that contrary to other reports, no significant differences in modified nucleoside levels were observed between urine samples from colorectal cancer patients and those from normal subjects. Trewyn and Grever (ref. 5) have pointed out that although certain species of tRNA may be hypermethylated in cancer cells (ref. 31), the degree of increased methylation of total tRNA is too low to be consistent

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with the high tRNA methyltransferase activity and capacity. A major Is altered tRNA metabolism a question which remains unresolved: fundamental aspect of neoplasia? Research on tRNA catabolites in urine and serum/plasma has concentrated on HPLC analysis of the modified nucleosides following isolation of the nucleosides by boronate gel affinity chromatography as the nucleosides are generally the major tRNA catabolic excretion products and are easily isolated by the boronate gel. However, immunoassays may be used more widely in the future to quantitate modified nucleosides in biological fluids, especially if the specificity and sensitivity can be achieved. Advancements i n the isolation, identification and measurement of modified nucleosides has been striking, and are now providing greater insights into the value of modified nucleosides as potential tumor markers. Early studies in which urinary modified nucleosides were found highIy elevated led to speculation that tumor tRNA was hypermethylated, and thus the modified nucleosides could be universal tumor markers. Development of these new research tools have brought insight into how modified nucleosides are excreted by healthy adults and children. With healthy adults the normal range of modified nucleoside excretion is very narrow, and it has been shown by Gehrke and Kuo that random urine samples can be utilized instead of 24-hour collections if nucleoside concentrations are expressed relative to creatinine. Adjustments must be made in the case of children, as the creatinine level correlates to body muscle mass. However, the excellent correlation of nucleoside excretion to age allows this adjustment to be made, resulting in a narrow normal excretion range (refs. 1, 32, 33). Trewyn and Grever (ref. 5 ) have provided an excellent review of urinary nucleosides and leukemia. They reviewed the available literature and discuss laboratory analyses, including methods, reference values, and multivariate analyses; clinical studies covering nonmalignant disease and infection, acute leukemia (childhood and adult) and chronic leukemias. They conclude that measurement of urinary nucleoside excretion offers a potential tool for monitoring disease activity in patients with ALL, CML, and perhaps CLL.

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They pointed out that additional work is necessary in following serial determinations of urinary nucleosides at frequent intervals in patients with different types of leukemia in order to assess the true value of these compounds as an accurate monitor of disease activity within the individual patient. They also observed that correlation of the nucleoside excretion pattern with ultimate clinical duration of complete remission is an important aspect that has not been adequately assessed. At this point, the major observations indicate that urinary nucleosides might serve as useful indicators for both prognosis and disease activity, although a significant amount of work remains to be done in order to ensure that these correlations are scientifically valid. Trewyn and Grever further point out that continuing investigation in the area of urinary nucleoside excretion provides a constant stimulus to understanding the biochemical changes which occur at the cellular level in leukemia, and that a clearer understanding of these cellular events may enhance understanding of the leukemogenic process itself. As Clark et al. discuss in Chapter 11, there is still disagreement concerning the source of the elevated RNA catabolites in patients with neoplasms. Although it is usually reported that the source and reason for the increased level of urinary RNA catabolites is increased turnover of tumor tRNA, Clark et al. and others are of the opinion that this increase is derived primarily from increased turnover of RNA in the host tissue (see Chapter 11, this volume). Salvatore et al. have focused especially on pseudouridine in blood serum as a biological marker of cancer, and in Chapter 7 discuss formation of the modified nucleosides, their measurement and normal levels in serum, and results obtained from the analysis of pseudouridine in serum of cancer patients. They report that in all groups of patients affected by tumors there was a definite and significant increase (as compared to normals) in blood pseudouridine levels, with the exception of the less advanced breast cancer groups; pseudouridine elevations were much greater for patients with lymphoma and leukemia: that there was a good correlation between tumor burden and/or spread of the tumor mass with blood pseudouridine levels; and that in the few cases where monitoring of neoplastic disease was correlated with blood

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pseudouridine levels, there was good correlation between the response to therapy and pseudouridine levels. They report this was the case for patients receiving chemotherapy and that underwent surgical treatment. Schoch’s group ha5 done much to clarify the origin of urinary RNA catabolites. They perceived a kind of basic stoichiometry in t h e pattern of the major modified excretion products, and thus began to screen the literature for relevant structural information. They discovered that modified nucleosides were distributed among rRNA, tRNA, mRNA and snRNA in proportions that were calculable. That ultimately enabled them to select specific urinary nucleosides for the whole-body turnover of each of the three miijor species of RNA; 7-methylguanine (i), N2,N2dimethylguanosine (ii), and pseudouridine (iii) are the degradation products from RNA turnover and can be used as markers for the wholebody metabolism of mRNA-cap, tRNA, and rRNA. The relative molar ratios of these molecules in serum is approximately 100:4.7:1.1 (see Chapter 13, this volume). Their approach opened a new way of looking at urinary RNA catabolites, which clearly adds a new dimension to future RNA catabolite investigations. Cimino et a!. report in Chapter 8 that evidence is accumulating where pseudouridine is the most highly and most frequently increased modified nucleoside in neoplastic patients, and that there is a good correlation between serum pseudouridine levels and progression of the neoplastic disease and the response to therapy. In Chapter 8 they describe increased pseudouridine levels in AKR mice and increased pseudouridine excretion by transformed cells. They also discuss enzymes involved in pseudouridine metabolism, modification of tRNAs from neoplastic cells and studies on tRNA primers for reverse transcriptase in tumor of retroviral origin. Still, the molecular basis for elevations of pseudouridine is unclear. After development of reliable methods for measuring modified nucleoside levels in physiological fluids, researchers began comparing urinary nucleoside levels of cancer patients with normal control subjects. Those data were presented in many cases in the form seen in

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Correlation of patient survival and urinary nucleoside levels. (From ref. 44 with permission of publisher)

Figure 1.1 as presented by Waalkes et al. (ref. 10). In this figure, the nucleosidelcreatinine ratios of four nucleosides from patients with colon cancer are compared with normal subjects. In many cases elevations were observed for persons with neoplastic disease. In 1982, Waalkes and Gehrke et al. (ref. 44) proposed a "composite score" approach for expressing urinary nucleoside values of patients with small cell carcinoma of the lung. As shown in Figure 1.2, patients with 3 to 5 elevated modified nucleosides had shorter survival times than patients with 0 to 2 nucleosides elevated. When the number

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of nucleosides elevated in concentration increased from 0-2 to 3-5 the A survival time dropped from about 24 months to 12 months. "composite score" approach has considerable merit in monitoring the course of the disease as the patient is receiving treatment. McEntire et al. (ref. 34) reported a study of serum nucleosides in 49 male lung cancer patients, 35 patients with other cancers and 48 patients hospitalized for non-neoplastic diseases. This study provided the most detailed serum nucleoside profiles reported to date, as 29 modified nucleoside peaks were normalized to an internal standard and analyzed by discriminant analysis, stepwise discriminant analysis, and principal components analysis. A model based on peaks selected by a stepwise discriminant procedure correctly classified 79% of the cancer It also demonstrated 84% and 75% of the non-cancer subjects. sensitivity and 79% specificity when comparing lung cancer to noncancer subjects, and 80% sensitivity and 55% specificity i n comparing lung cancer to other cancers. The nucleoside peaks having the greatest influence on the models varied dependent on the subgroups compared, confirming the importance of quantifying a wide array of nucleosides. Using principal components analysis, 65% of the cancer patients and 79% of the non-cancer patients were correctly classified and the modeling power of each of the 29 nucleosides was also determined. These data support and expand previous studies which reported the utility of measuring modified nucleoside levels in serum, and show that precise measurement of an array of 29 modified nucleosides in serum by HPLCUV with subsequent data modeling may provide a clinically useful approach to patient classification in diagnosis and subsequent therapeutic monitoring. Chheda et al. (ref. 35) recently made an evaluation of 5 carbamoylmethyluridine (ncm5U) as an indicator of tumor burden in lung cancer patients and found that the levels of ncm5U were elevated i n the urine and serum of non-small cell lung cancer patients when compared to the levels found in normal subjects (p = < 0.001). Significantly elevated levels of ncm5U were found in the urine of 17 of 18 (98%) of the patients. These investigations support the use of ncm5U as a monitor of tumor load since combined urinary/serum levels reflected advanced malignancy.

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In collaborative programs with Professor F. Salvatore and his research group at the University of Naples Medical school, Dr. Edith Mitchell in the Department of Oncology, University of Missouri Medical School, and Dr. John McEntire of the Cancer Research Center, Columbia, MO, we collected 94 samples of serum from normal healthy donors and 47 serum samples from non-cancer male patients. The normal healthy population consisted of 51 males and 43 females ranging in age from 19 to 84. Thirteen serum modified nucleosides and creatinine were quantified and the data are presented in the following chapter. Briefly, the narrow distribution (RSD, %) of each nucleoside in the 94 samples was essentially the same whether the data were expressed as pmol/ml or as the nucleoside/creatinine ratio. This indicates a stringently controlled metabolic rate of nucleic acids for healthy subjects. There is no age and sex dependency for adults of any of the nucleosides studied. Thirteen serum modified nucleosides in patients with a number of diseases other than cancer (DOTC) were also investigated. This study included 47 males with ages ranging from 27 to 83. The nucleoside values for the DOTC patients were essentially the same as for the n or m a 1s . Sixteen urinary nucleosides and creatinine were measured in 24 hour collections of urine from 18 normal healthy donors (7 males, 11 females, ages 25 to 50). A narrow distribution of each nucleoside was again observed i n the urine of normal healthy subjects as for serum in healthy subjects. In further collaboration with Professor F. Salvatore's group at the University of Naples, serum from pretreatment leukemia and lymphoma patients were collected and analyzed. Brief preliminary results are presented as bar graphs i n the following chapter. Comparisons of the normal serum nucleoside levels to the levels found i n acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, and chronic myeloid leukemia (CML) were made, as was a comparison of the normal serum nucleoside levels to the levels found in Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). We found that the level of modified nucleosides from the patients in all types of leukemia and lymphoma are significantly

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higher than for the normal values, and acute lymphocytic leukemia patients have much higher levels than patients with other types of leukemia and lymphoma. This indicates the excellent diagnostic value of modified nucleosides for leukemia and lymphoma. The preliminary data also shows that the modified nucleoside profiles of some leukemias are different from the other leukemia types. Thus, preliminary studies on serum nucleosides as potential biological markers for small cell lung carcinoma, leukemias and lymphomas were achieved. Some significant correlations were noted between the levels and profiles of serum nucleosides and different neoplasias.

SUMMARY Much research has been conducted with the general goal of identifying a component(s) of a physiological fluid which would serve as a tumor o r cancer marker. The modified nucleosides resulting from altered t R N A metabolism are i m p o r t a n t as potentially useful "biochemical sentinels" in the diagnosis, prognosis, and evaluation of therapy and monitoring the disease activity in cancer. The term "tumor marker" as coined by Dr. Morton K. Schwartz of the Sloan Kettering Institute refers to some unique metabolic product(s) or unique component(s) of malignant cells which can be measured in body fluids. As pointed out by Borek and others, the criteria of an effective tumor marker are numerous; it should be specific for malignancy; it should provide a minimum of false-positives and falsenegatives; it should indicate the extensiveness of the malignancy and it should preferably diminish or hopefully disappear after effective therapy. There have been indications for more than 30 years that cancer patients excrete elevated levels of modified nucleosides. The origin of these compounds was obscure until the discovery of the modification of transfer RNA. However, the events responsible f o r the increased excretion of modified nucleosides by cancer patients remain unclear, with increased tRNA turnover considered as the predominant source, and cell death and increased turnover of RNA in the host tissue also considered by various investigators. 1.4

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Trewyn and Grever have pointed out that although certain species of tRNA may be hypermethylated in cancer cells, the degree of increased methylation of total tRNA is too low to be consistent with the high tRNA methyltransferase activity and capacity. A major question which remains unresolved: Is altered tRNA metabolism a fundamental aspect and/or consequence of neoplasia? Research in our laboratories on tRNA catabolites in urine and serum/plasma has centered on the development of HPLC separation and UV diode array detection of the modified nucleosides following selective isolation of the nucleosides by boronate gel affinity chromatography as the nucleosides are generally t h e major tRNA catabolic excretion products and are easily isolated by the boronate gel. However, immunoassays may be used more widely in the future to quantitate modified nucleosides in biological fluids if the specificity and sensitivity of the method can be improved. Tjaden (ref. 3 6 ) has pointed out that the analytical and preparative separation of specific compounds in complex samples like physiological fluids and cell extracts is of fundamental importance in biomedical research. Nucleotides, nucleosides and their bases are not only the essential constituents of nucleic acids, but also of other structures important for the proper functioning of cells. Since physiological fluid levels of nucleosides are dependent on the metabolic state of cells, nucleoside profiles might be used in monitoring the progression of disease or the therapeutic effects of drugs. Various analytical methods for the determination of these compounds have been developed, but HPLC-UV is one of the most popular techniques in this respect, since it combines the high selectivity of the separation method with sensitivity of detection (ref. 37). With respect to molecular mass and to polarity, a wide array (-30) of modified nucleosides can be measured by HPLC simultaneously, making this technique a powerful analytical tool. In the past few years, advancements i n the isolation, identification and measurement of modified nucleosides has been striking, and are now providing greater insights into the value of modified nucleosides as potential tumor markers. Early studies in which urinary modified nucleosides were found highly elevated led to speculation that tumor

c 34 tRNA was hypermethylated, and thus the modified nucleosides could be universal tumor markers. Our development of these analytical-chromatographic methods (ref. 37) has brought insight into how modified nucleosides are excreted by healthy adults and children. With healthy adults, the normal range of modified nucleoside excretion is very narrow, and we have shown that random urine samples can be utilized instead of 24-hour collections if nucleoside concentrations are expressed relative to creatinine. In normal patients the excretion level of the modified bases was demonstrated as remarkably constant. Adjustments must be make in the case of children, as the creatinine level correlates to body muscle mass. However, the excellent correlation of nucleoside excretion to age allows this adjustment to be made, resulting in a narrow normal excretion range. An area that seems promising for biologic markers is that of leukemia (ref. 5) research. Trewyn and Grever have provided an excellent review of urinary nucleosides and leukemia. They reviewed the available literature, and discuss laboratory analyses, including methods, reference values, and multivariate analyses; clinical studies covering nonmalignant disease and infection, acute leukemia (childhood and adult) and chronic leukemias. They conclude that measurement of urinary nucleoside excretion offers a potential tool for monitoring disease activity in patients with ALL, CML, and perhaps CLL. In addition, they pointed out that further work is necessary in following serial determinations of urinary nucleosides at frequent intervals in patients with different types of leukemia in order to assess the true value of these compounds as an accurate monitor of disease activity within the individual patient. They also observed that correlation of the nucleoside excretion pattern with ultimate clinical duration of complete remission is an important aspect that has not been adequately assessed. At this point, the major observations indicate that urinary and serum nucleosides might serve as useful indicators for both prognosis and disease activity, although a significant amount of serial studies remain to be done in order to ensure that these correlations are scientifically valid.

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Further, continuing investigation in the area of urinary nucleoside excretion provides a constant stimulus to understanding the biochemical changes which occur at the cellular level in leukemia, and that a clearer understanding of these cellular events may enhance understanding of the leukemogenic process itself. During the last two years we have improved and extensively validated the quantitation of ribonucleosides in biological samples (see Chapters 1, and 5, Part A; and Chapter 2 , Part C). This technology represents a significant advancement over t h e methods that we reported earlier (refs. 38, 39). The precision, speed, sensitivity and ruggedness of our methods are well suited for use in clinical research applications. With the described chromatography protocols, twenty known nucleosides in urine or serum and more than ten unidentified nucleosides can be measured in a single 35 minute chromatographic run. The precision and ruggedness of the method was ensured with the introduction of a new internal standard, 3-methyluridine (m3U), which is added to the urine or serum sample before prechromatography treatment. Also, the accuracy of the method was improved by employing a UV diode-array detector and multi-wavelength quantitation protocols. In our laboratory this nucleoside methodology has been applied on approximately 500 human serum samples, and 200 urine samples with consistent satisfactory results. As presented in the following chapter by Kuo and Gehrke, thirteen human serum nucleoside levels and 17 human urinary nucleoside levels were established on analysis of a large number of samples from a normal population. In addition, preliminary studies on serum nucleosides as potential biological markers for small cell carcinoma, leukemias and lymphomas were achieved. Some significant correlations were noted between the levels and profiles of serum nucleosides and different neoplasias. In a number of collaborative investigations (ref. 40-43) we have extended HPLC-UV analysis to the quantitative analysis of the complete tRNA molecule, with special emphasis on the extensive and complex modifications present in tRNA (see Chapter 1, Part A), improved and interfaced characterization methods (HPLC-UV, MS, NMR, and FT-IR) for elucidating the structures of unknown modified nucleosides (see

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Chapter 5, Part A), developed methods for quantitatively studying the cap structures of messenger RNA (see Chapter 8, Part A), and to a rapid and accurate methodology for studying methylation of DNA (see Chapter 10, Part B). In this research we found new modifications in tRNAs, specifically in position 64 of yeast methionine initiator tRNA, in which 0-prib of u r a n o sy 1- ( 1" - 2') -ad e n o sine - 5 " -phosphate is 1inked by a 3 ' ,5 ' phosphodiester bond to G at position 65 (ref. 40). Also, a new major modified nucleoside (C*) has been identified as om5C in canine serum (see Chapter 5 , Part A); the study of antisuppressor mutations and sulfur-carrying nucleosides in transfer RNAs of schizosaccharomyces pombe has been published (ref. 41), and this technology has been applied to the identification and measurement of polynuclear carcinogen-ribonucleoside adducts in the urine of fish and rat (Chapter 2, Part C). This methodology has also been used to investigate codon discrimination and anticodon structural context (ref. 42), and the finding of 5-carboxymethylaminomethyluridine in the anticodon of yeast mitochondria1 tRNAs recognizing two-codon families ending in a purine (ref. 43). The broad applicability of RPLC-UV real time diode array analysis was demonstrated by the analysis of nucleosides in human plasma, whole blood, and other biological samples. The measurement and simultaneous detection of an array of nucleosides in complex biological matricies has been demonstrated and widely applied. These new nucleoside chromatography research tools will serve to advance biochemical and biomedical investigations, and present new research approaches to further studies in molecular biology.

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