BLOCHEMICAL MARKERS OR ENZYME CHANGES THAT MAY PRESAGE THE PRESENCE OF CANCER CONTRACT NUMBER NO1-CB-43902 Progress Report for the period July 1, 1975 to January 31, 1976. "J. Lederberg 0 Professor of Genetic Principal Investigator BIOCHEMICAL MARKERS OR ENZYME CHANGES THAT MAY PRESAGE THE PRESENCE OF CANCER Contract Number NO1-CB-43902 Progress Report for the Period July 1, 1975 to January 31, 1976 Biochemical Markers or Enzyme Changes that May Presage the Presence of Cancer This report discusses progress made during the above period and continuing to date on the two areas of emphasis of our recent research: A. Development and application of an analytical method for quantitation of urinary polyamine levels. B. Screening of urine for metabolites which might presage the presence of cancer. A. Development of an Analytical Method for the Quantitation of Urinary Polyamine Levels. A sensitive and specific method using mass fragmentography for the analysis of the polyamines putrescine, cadaverine, spermidine and spermine has been developed, along with a method of synthesis for their deuterated analogs. The procedure involves addition of a known amount of a standard solution of deuterated analogs to the urine, followed by overnight acid hydrolysis, butanol extraction and ion exchange chromatography on a strongly cationic ion exchange resin. These procedures have been described in detail in previous reports. The polyamine extract is triflouroacetylated, and polyamine quantitation is achieved by measuring peak height ratios of specific ions characteristic of the triflouroacetyl derivatives of the indigenous and deuterated polyamines, m/e 126 and m/e 154 being characteristic of the derivative of the indigenous material, and 128 and 156 being characteristic of the derivative of the deuterated analog. After injection of the samples into the GC/MS system, approximately one-half hour is required for analysis, followed by five minutes for processing the analytical data. The concentrations of indigenous polyamines are expressed in mg/100 ml at the end of the time required for processing. During the actual GC/MS runs, the computer monitors the specific ions (126, 128, 154 and 156) characteristic of the materials being analyzed, and at termination of the runs, the total ion current (Fig. I) is printed out at a CalComp plotter. During processing of the analytical data, the contributions of the individual specific ions to the total ion current are separated and displayed on a T.V.Monitor along with background subtraction (Fig. II), and the finding of the peak's maximum (Fig. III). Before the actual urine analysis, several quantitative mixtures of pure deuterated polyamines and pure non-deuterated polyamines are made up and derivatized. These mixtures are analyzed by GC/MS and processed to determine the specific ion ratios for each polyamine. These ratios are then plotted versus relative concentrations of the nondeuterated and deuterated polyamines to establish calibration curves for the individual polyamines (Fig. IV). During the actual urine analysis, the calibration curves are used to determine the relative concentration of the non-deuterated indigenous materials, from the specific ion ratios determined. The concentrations of the polyamines in the urine can then be determined. Our intent in pursuing this study is to determine if the polyamines can be used as markers for the early detection of prostatic cancer. Prostatic cancer was chosen, because the highest concentrations of poly- amines in the human body can be found in the male prostate gland. After the method had been developed, it was applied to clinical analysis. To date, sixteen prostatic cancer urines, eight benign prostatic hypertrophy urines and nine control urines have been run. One Hodgkins urine and one breast cancer urine have also been run. Three BPH urines, one prostatic cancer urine and three control urines have yet to be run. We have yet to process the analytical data. The clinical data on all of these patients, including controls, is included in this report. More extensive clinical data is available for inspection. TABLE I. Lab. No. 229 238 239 302 324 329 330 336 337 340 343 344 345 347 348 349 351 354 356c 362 363 1-74 2-74 3-74 4-75 5-75 6-75 7-75 8-75 9-75 10-75 11-75 12-75 RO OnN AU HEWN Disease Ca. Prostate Normal Normal Ca. Prostate Hodgkins Ca. Prostate Grade II Ca. Prostate Grade I BPH BPH BPH BPH BPH BPH BPH BPH BPH BPH Ca Prostate Grade II BPH Ca. Prostate Grade IIL Ca. Prostate Grade II Ca. Prostate Grade II Ca. Prostate Grade III Ca. Prostate Grade III Ca. Prostate Grade III Ca. Prostate Grade I Ca. Prostate Grade III Ca. Prostate Breast Ca. Ca. Prostate Ca. Prostate Ca. Prostate Ca. Prostate Control Control Control Control Control Control Control Control Control Control Age 51 70 58 29 69 61 56 61 64 62 62 71 71 51 51 51 52 64 74 63 64 65 56 65 54 66 50 50 51 61 ? 39 33 39 35 27 26 41 43 35 ? Urine Volume 1100 cc 1410 ec (12 hr) 1840 (12 hr) 1610 cc 632 ce (12 hr) 1135 ce 2370 cc 1300 ce (12 hr) 1790 cc 1430 ce (12 hr) 990 ce (8 hr) 745 ec 1250 ce (16 hr) 372 cc (8 hr) 900 cc (8 hr) 590 ec (8 hr) 3220 ce 620 ce (8 hr) 1425 ce 1550 cc 970 ec (12 hr) 720 cc (12 hr) 560 ec (12 hr) 440 ce (12 hr) 525 ec (12 hr) 620 cc (16 hr) 407 ce (12 hr) 1240 ce 695 ce (12 hr) 2140 cc 475 ec (12 hr) 1050 ce (12 hr) 837 cc 1760 ce 1460 cc 1758 ce 930 cc 1020 cc 1248 cc 2030 cc 1430 cc 1330 cc Summary of Data Available for Polyamine Analysis Chemistry x OM MMM MM OM MOM OM OR OM ™ ” bd a wm OM OM OM MMM MM MM OM OM OM Gc/MS x mM ” MM MO OM MO OOM “ x MM OM OM “ mo MMMM OM OM Final Anal. B. Screening of Urine for Metabolites Which Might Presage the Presence of Cancer During the period covered by this report, we have essentially completed the GC/MS profiles on the six fractions of the organic constituents of urine from patients with a variety of cancers. Subsequent preliminary computer analysis of the data is also essentially complete. The status of each of the samples provided to us by Dr. Waalkes of the National Cancer Institute is summarized in Table II. We are currently trying to obtain from Dr. Waalkes, now at Johns Hopkins, the patient histories corresponding to these samples. As before, the urine from each of these patients was fractionated into: (1) an acidic and neutral fraction; (2) an amino acid fraction and (3) a sugar fraction. The acidic and neutral fraction was divided into two equal portions, one of which was methylated with diazomethane (D-OME) while the other was silylated with BSTFA + 1% TMCS (D-TMS). The sugar fraction was derivatized to the TMS derivative (S-TMS) with TRI-SIL-Z. The amino acid fraction was also divided into two equal portions with one portion silylated with BSTFA + 1% TMCS (E-TMS) and the other converted to N-TFA-O-n-butyl derivative (E-TAB). Details of the procedure have been presented in previous reports. Each of the six fractions of each urine was then analyzed by the GC/MS/Computer system. Each fraction yields about 600 complete mass spectra These spectra are processed by a computer program, called "CLEANUP", which is designed to detect components and remove from the spectrum of each component interference from background, column bleed and overlapping components. This procedure yields spectra which are much more characteristic of the spectra of pure compounds than are the raw data. Each fraction may yield from 30-60 component mass spectra, a considerable data reduction from the original 600 spectra, many of which are background. We have assembled libraries of mass spectra of known compounds by dividing an available collection of over 3000 spectra of compounds of biological interest into subclasses corresponding to the chemical fractions isolated in the above procedure. The appropriate library is searched for the spectrum of each component detected by CLEANUP. Spectra of components which were not matched to the library are examined further in collaboration with the NIH supported DENDRAL project for computer-assisted structure elucidation. Preliminary manual examination of the above data has revealed large amounts of B-aminoisobutyric acid (BAIB) excreted in the urines of three of the six patients with lung cancer. We have previously reported the association of increased urinary BAIB excretion with several lukemic, bladder, prostatic and lymphoma forms of cancer. Although precise quantita- tion is not yet available (see below) for the other samples (Table II) which do not show such large amounts of BAIB, the frequency with which this material appears in grossly elevated amounts in the samples we have examined is remarkable. Table II. Status of Analysis of Organic Constituents of Urines of Patients with Various Cancers. Breast Cancer Chemistry GC/MS Analysis 154 (007) Completed Completed Computer Analysis Completed 383(152) 282 (084) 953 (432) 751 (343) 448 (193) Lung Cancer 306 482 511 586 639 779 Pancreas Cancer 314 (110) 387 (156) 532 (244) 668 (314) 1120(508) 752 (349) Colon Cancer 533 (245) 623(300) 993 (456) 1585 (621) 1586 (622) 1799 (676) Completed tf ft a Completed "W except D-TMS except D-TMS Completed Completed Completed Completed Completed Completed Complted Compited Completed " except D-TMS except D-TMS Completed Completed Completed Completed Completed Completed Completed except S-TMS & E-TAB Completed " " " wt LA Completed i except Except Except except except except except except except except D-TMS D-TMS E-TMS E-TMS E-TMS E-TMS E-TAB & E-TMS E-TAB & E-TMS E-TAB & E-TMS - E-TAB,E-TMS & S-TMS Several previously unencountered and currently unidentified components have been detected in some samples, for example, two new components in the spectra of several of the lung cancer samples. Because these may be artifacts (e.g., drugs or drug metabolites not covered in our current libraries) any more definitive statement must await the patient histories. Results of the past 18 months have led us to pursue two lines of activity for the remaining period of this grant, in addition to finishing the few analyses which remain in the preliminary collection and analysis of data (Table II). 1. Precise quantitation of BAIB. We can evaluate the utility of BAIB as a potential diagnostic marker only when several additional experiments are completed. The first is precise quantitation of BAIB in sets of samples where it was detected in some patients at high levels and comparison of these data with approximate controls. To this end, we have begun refining our current method for the quantitation of BAIB in urine by mass fragmentography ("The Quantitation of B-Aminoisobutyric Acid in Urine by Mass Fragmentography", W. E. Pereira, R. E. Summons, W. E. Reynolds, T. C. Rindfleisch, and A. M. Duffield, Clinica Chimica Acta 49, 401-406, 1973). This method, utilizing a Tabsorb GC column and the GC/MS/ computer to monitor masses 153 and 182 of the N-TFA-O-n-butyl ester of BAIB, suffers from interference of masses of isoleucine, which elutes from the GC with the same relative retention index. We have determined (by high resolution mass spectrometry provided by our DENDRAL collaborators) that the mass 74 ion of BAIB-N-TFA-O-n-butyl ester is unique to BAIB, possesses composition C3Hg0) and most probably represents the portion of the molecule circled in structure 1. We will synthesize the deuterium labeled analog 2 and monitor m/e 74 with respect to m/e 77 (from the same fragmentation of 2; 2 added in known quantities) to quantitate BAIB. g i CF ,-C-NH-CH, n-Bu CF.,-C-NH-CH, n-Bu + 2H + 2H m/e 74 m/e 77 i 2 2. Detailed Intercomparison of Samples. We must examine the excretion profiles of the patients in more detail. Manual examination can catch only the grosser abnormalities, because there are far too many data for more careful comparison of results of one sample with a previous history of results. We plan to develop the computer programs necessary to automate this procedure. The concepts are straightforward and several of the programs are largely modifications of existing software. The goal of this effort is to provide the chemist a summary report on the similarities and dissimilarities among the organic constituents of urines of selected sets of patients. It must be flexible enough to compare patients with the same or different cancers or either with controls, and to compare a patient or patients with a more comprehensive history of components detected in any previous analysis independent of knowledge of the structure of the component. , Briefly, these programs will begin after CLEANUP and library search are completed. The relative retention index of each component, determined from hydrocarbon standards added to the data, is calculated. Each set of spectra is then compared to a "local" library of spectra (from one or more related patients or a more comprehensive set) where the matching criteria are retention indexes and similarity of spectra. The hydrocarbon standards provide a means for semi-quantitative estimation of the relative amounts of each component. NANCY'S #344 BPH 01-DEC-75 ETE X1000 ET303 TTT Bue 28a 78 Pe ee TT PTT Ty Boe —— T Zoe FIGURE I - a a EE aml A RR PTT mS ft en ennneene yee — _ . MS 15@ cag 144 = 12@89SCKGROUND APPROXIMATION: CABAVERINE: MASS FIGURE II ea 15a 128 THRESHOLDED PEAKS 4 AREAS: CABAVER iQ SB CETTE Tre INE: YY) a S FIGURE III 10 CALIBRATION CurRVE + CADAVERIWE. TT i. o-+ 2? OF of 4. v SPEciFIG low RATIOS pie 1ab/mi 12? o.3 05 06 2&7 own oO Of OF OF OF OF OF OF OF Cf Ko RATIO «OF wow DEUTERATED TO DEUTERATED MATERIAL. AI Wqanols