RFP TITLE:

BIOCHEMICAL MARKERS THAT MAY PRESAGE

THE PRESENCE OF CANCER

RFP NO.

NCI-CB-74-29 (PROJECT NO. CB~43902-S)

AMENDED PROPOSAL (JUNE 18, 1974)

SUBMITTED BY

THE BOARD OF TRUSTEES OF THE LELAND STANFORD, JR. UNIVERSITY

Quine hy

rq
Joshua Lederberg, Principal’ Investigator
Department of Genetics a .

Stanford University Medical School
RFP No. NCI-CB-74-29

INTRODUCTION

The aim of this proposal is to use gas chromatography (GC) in
conjunction with computerized mass spectrometry (MS), to investigate
the presence of metabolites and metabolic changes occurring in
biological fluid that may presage the presence of cancer.

In writing up this amended proposal we have taken into consideration
the valuable comments and suggestions put forth by the site visit
committee. Their suggestion accords with our own criticisms to narrow
the emphasis to the screening of small selected groups of patients
with specific types of cancer. In addition we briefly describe these
segments of our research protocols which we modified after discussion
with the site visit committee.

We propose using our existing analytical procedures, and also by
developing new methodology, to begin studying limited populations of
patients with the following types of cancer:

(a) prostatic cancer
(b) bladder cancer (multiple, recurrent, stage A carcinoma)
(c) diagnosed leukemia

(c) non-Hodgkin's lymphomas

In addition we are purusing, at the suggestion of the site visit
committee, ways of obtaining urine specimens from a relatively small
number of adult patients with Hodgkin's Disease and others with cancer
of the pancreas. As these samples materialize they will be screened
for their metabolic content.

Specifically we intend to screen 10-12 urine samples from patients
with each of these cancers and for a suitable number of controls (see
below) for biochemical markers indicative of the disease state. In
addition we will demonstrate the methodology required for the sub-nanogram
quantitation of urine polyamine levels in order that this technique will
be available for exploitation in subsequent years of this contract.

We also intend to monitor the urinary beta~aminoisobutyric acid levels
in children with diagnosed leukemias and non-Hodgkin's lymphomas.
RFP No. NCI-CB-74-29

AVAILABILITY OF BIOLOGICAL -SAMPLES

Biological specimens will be made available to us by the Clinical
Research Center and the Urology Clinic of the Stanford University
Medical School through cooperation of Dr. W. Fair (Associate Professor
of Surgery), and Dr. T. Long (Children's Hospital, Oncology Division,
Stanford University).

METHODOLOGY

The gas chromatograph-mass spectrometer (GC-MS) system will be
primarily used to separate and identify biological metabolites present
in the urine of cancer patients. It is reasonable to assume that
some metabolites that presage the presence of cancer may be present in
extremely minute amounts (picogram levels). For the purpose of their
detection and quantitation we propose using the technique of mass
fragmentography (see original proposal). In the examples of urinary
polyamine levels, where the sensitivity of detection is crucial, we intend
to develop new analytic techniques capable of quantitating these compounds
below the nanogram level.

SPECIFIC AIMS AND OBJECTIVES

An ancillary objective, in addition to looking at metabolic
profiles of cancer patients (introduction a,b,c,d), will be directed
towards a quantitative investigation of the following two biochemical
markers that have been reported to be present at elevated concentrations
in various types of neoplastic disease. This will involve accurate
quantitation of the following urinary metabolites:

1) Polyamines in prostatic cancer

2) Beta aminoisobutyric acid (BAIB) in leukemia (see original
proposal). .

The development of more sensitive analytic methods is aimed at
answering questions of the mechanisms of the increases in polyamines
noted by Dr. Fair. This would entail comparisons of prostatic fluid,
voided urine and bladder urine from his patients. If tissue.samples
of 100 mgs or less can be assayed, surgical specimens can be examined--
larger samples interfere with necessary histopathological scrutiny.

SCREENING URINE SAMPLES FOR BIOCHEMICAL MARKERS

 

Urine from patients afflicted with the four types of cancer
listed in the introduction will be screened by GC/MS procedures as
detailed in our formal proposal-
RIE WOs. WNULTGD"/4~-243

Bladder cancer, specifically multiple, recurrent, stage A .
carcinoma, presents a special opportunity for metabolic analysis. The
‘multiple recurrent lesions suggest a systemic basis for the disease;
hence a theoretical rationale for metabolic screening and perhaps
genetic etiologies. The Urology Department sees almost 50 cases per
year of this condition and we foresee no difficulty in collecting about
12 for a one-year protocol. The sample collections and protocol
regimens will correspond to those in the prostatic cancer series, who
will also serve as adjunct controls. Similar comments apply to
"normal" controls. (One must keep in mind that most normal middle aged
men are subject to some stage of neoplastic disease of the prostate).

For this condition we will place special emphasis on family studies.
In a preliminary search for signature substances, we will scan siblings,
spouse and off-spring of the propositus where available. If such
substances are identified they will be systematically tracked through
pedigrees by conventional genetic methods.

CLINICAL PROTOCOL FOR SCREENING URINE SAMPLES FOR BIOCHEMICAL MARKERS

(a) Collection of samples. All urine samples will be 24 hour
collections. The urine will be collected in sterilized glass bottles
under toluene and frozen immediately until ready for use.

 

(b) Prestudy regimen. Urine specimens will be obtained from
patients who have discontinued all medication for at least 48 hours
prior to collection. Dietary controls would not be possible on non-
hospitalized patients. The patients selected will be free of fevers,
emaciation, anemia, uremia or malaise. Where possible all patients will
be surgically staged. In other instances clinical records will be used
to provide data on the extent of the disease. The study groups for
each of the four categories of disease will consist of between 10 and
12 patients.

(c) Controls. These will be matched according to age, weight,
‘sex and smoking habits.

(d) Intervals of Study. 24-hour urine collections will be
repeated twice at suitable intervals (30, 60 day) following the initial

collection.
1) Polyamines in Prostatic Cancer

Carcinoma of the prostate ranks second among the causes of male
cancer deaths in the United States (1). At present there is no accurate
method for early detection of prostatic cancer. It has been found that
the polyamines putrescine [H N- (CH, ) ‘ ], spermidine [NHg- (CH 5)yNH- (CH2),°NHo]
and spermine [NHo* (CH>)3°NH- CH) 42NH- (CAly) 3°NHy] have a strong affinity
for the nucleic acids RNA and DNA (2). Increased levels of polyamines
RFP No. NCI-~CB-74~29

have been reported in rapidly growing neoplastic tissue (3) and in

the urine of patients with different types of cancer (4). Since the
highest concentrations of the polyamines found in the human body occur
in the prostate (5), it would be significant to find if a correlation
exists between carcinoma of the prostate and urinary excretion levels
of the polyamines, This study would provide information concerning the
urinary concentration of pelyamines which might then be related to the
presence of carcinoma of the prostate.

Initial studies by Dr. W. Fair's group in the Department of Urology,
Stanford University Medical School, have shown (using paper electrophoresis)
that urinary levels of spermidine appear to be significantly elevated
in many patients with prostatic neoplasms. Following our discussion
with the site visit committee we propose to expand on the initial
investigation of Dr. Fair by developing a methodology for the sub-nanogram
quantitation of the polyamines, putrescine, spermidine and spermine.

If this methodology can be developed and tuned for optimum sensitivity
we will be in a position to resolve the conflicting evidence present in
the literature on the efficacy of urinary polyamine levels as markers
for the detection of cancer.

Preliminary studies we have made on the synthetic TFA-polyamines
have shown that the chemical ionization mass spectra of these compounds
are ideally suited for mass fragmentography. The electron impact
spectra of these compounds do not show molecular ions, but show extensive
fragmentation resulting in generally low abundance ions, which do not
result in optimum sensitivity using mass fragmentography. On the other
hand, these polyamines are good proton acceptors and their TFA derivatives
give abundant quasi-molecular ions (M+ 1), in the chemical ionization
mode making them an ideal derivative for quantitation by mass fragmentography.
Since the polyamine levels in urine are extremely low (< 1-2 mg/24 hrs.)
we feel that chemical ionization-mass fragmentography is the most
promising method available for the quantitation of these biochemical
markers. To this end we have approached instrument manufacturers for
time to use their instruments to verify the feasibility of our intended
assay as we do not have access to a chemical ionization mass spectrometer.
In the first year we wculd demonstrate the feasibility of a chemical
ionization-mass fragmentography methodology for sub-nanogram quantitation
of putrescine, spermidine and spermine. We would then begin to apply.
the technology to the routine determination of urinary polyamine levels
in patients with prostate cancer and other carcinomas and for assaying
polyamine levels in tissue obtained from surgical procedures.

CLINICAL PROTOCOL FOR Il VESTIGATION OF POLYAMINES IN PROSTATIC CARCINOMA
(a) Collection of samples. All urine samples will be 24-hour

collections. The urine will be collected in sterilized glass bottles
under toluene, and frozen immediately until ready for assay.
Rep No. NCI-CB-/4-29

(b) Prestudy regimen. Urine specimens will be obtained from
patients who have discontinued all medication for at least 48 hours
prior to collection. Dietary controls would not be possible on non-
hospitalized patients. The patients selected will be free from fevers,
emaciation, anemia, uremia or malaise. Alli patients chosen for the
study will be surgically staged, so that the extent of the disease is
accurately determined. The study group will consist of 10-12 patients
with diagnosed prostatic carcinoma.

(c}) Controls. Controls obtained from out-patient clinic volunteers
will be matched according to age, weight, and smoking habits. In addition
samples from other urology patients will serve as adjunct controls.

(d) Intervals of study. A 24-hour urine collection will be made
on each patient prior to surgical staging and two more specimens. —
collected after the patient undergoes surgery. The patient will be
followed up in the Urology Clinic and 24-hour urine specimens obtained
at appropriate intervals.

2) Beta Aminoisobutyric Acid (BAIB) in Leukemia and Non-Hodgkin's Lymphomas

During 2a preliminary screening program of the urinary metabolites
of children with diagnosed leukemias, we observed several cases in
which the levels of BAIB were substantially elevated (> 1g/24 hrs).
Since existing procedures for the urinary analysis of BAIB were not
specific enough, we developed a sensitive method for quantitation of BAIB
in urine by Mass Fragmentography (6). BAIB has been shown to be one of
the end products of DNA catabolism, and thymine is its principal precursor (7).
In conditions such as neoplastic diseases, one might expect a relationship
to exist between the excretion levels of BAIB and the DNA metabolic process.

We propose measuring BAIB levels, as an indicator of disease activity
in the urine of children with newly diagnosed leukemias and non-Hodgkin's
lymphomas. Details of the experimental protocols will be found in our
original contract proposal.

CLINICAL PROTOCOL FOR INVESTIGATION OF BAIB IN LEUKEMIA AND NON-HODGKIN'S
LYMPHOMAS

{a) Collection of samples. All urine specimens will be 24-hour
collections. The urine will be collected in sterilized glass bottles
under toluene, and frozen immediately until ready for assay.

 

(b) Pre-study regimen. Urine specimens will be obtained from
patients who have discontinued all medication for at least 48 hours
prior to collection. A record of the patients diet will also be obtained
from the dietician at Children's Hospital, Stanford University. The
patients selected for study will.vary from two to fifteen years of age.
Care will be exercised to select patients who do not have fevers,
ANA SENN ee ABN LT aS

emaciation, anemia, uremia or malaise. Groups of 10 to 12 patients
with newly diagnosed leukemia, and 10 to 12 patients with non-Hodgkin's
lymphomas will be used for the study.

(c) Controls. Controls for the two groups to be studied will be
of similar age, sex and weight and will be chosen from patients in the
Children's Hospital confined for treatment of other than disease.

Urine specimens from the control patients will be provided by the Chief
Resident of the hospital at Stanford University Medical School.

(d) Intervals of study. All urine specimens of patients in the
two study groups, will be collected at intervals of 30 and 60 days.
following the initial collection.

ANCILLARY STUDIES ON TISSUE SAMPLES AND CELL CULTURES

There are two principal rationales for seeking biochemical signatures
of cancer;

1) a constitutional -- genetic or environmental ——- systemic
predisposition, and

2) a direct metabolic effect of the incipient tumor mass itself.

In the foregoing presentation, 1) is represented by the planned
studies on bladder cancer; 2) on prostatic.

Since the metabolic output of an incipient tumor, unless amplified
by systemic control mechanisms, will be diluted in the whole body space,
it may be difficult to identify in the overall urinary output or in plasma
solution.

We therefore plan to include some pilot studies along the following
lines:

a) displacement of albumin-bound metabolites, as has been noted
as a side-effect of uricosiric drugs, and

b) scanning tissue specimens (surgical, biopsy, and cell cultures)
for signature substances that may be expected to be found at
higher concentrations in sicu than in the body fluid. (Once
such substances are identified, they can be sought more
assiduously in the body fluid samples.)
RFP No. NCI-C3B-74-29

In this connection, Professor Hilary Koprowski of the Wistar Institute,
University of Pennsylvania, has offered to provide hybrid cell lines
which are segregating for a single human chromosome that influences the
neoplastic~transformation characteristic.

These studies are not expected to take a significant part of our
effort under the present contract; but if successful they may be the basis
of requests for support for larger scale efforts.

It should be noted that the epidemiological and experimental
designs presented hereinabove are tentative predictions. Rigorous, detailed
prospective efforts will be needed to substantiate the validity of any
preliminary findings that may emerge from the presently contemplated effort;
and these can be designed only on the basis of the information we are now
seeking. oo
1.

2.

3.

4.

BLE WW. NULWMGD-s/ 4-247

REFERENCES

American Cancer Society (1970). Cancer Facts and Figures;
Biostatistics of the United States.

Tabor, H. and Tabor, C. W. "Spermidine, Spermine and Related
Amines." Pharmacol. Rev. 16: 245 (1964).

‘Russell, D. H., Levy, C. C., Schimpff, S. C., and Hawk, I. A.,

“Urinary Polyamines in Cancer Patients." Cancer Res. 31, 1555 (1971).

Denton, M. D., Glazer, H. S., Walle, T., Zellner, D. C., and
Smith, F. G. In Polyamines: Their Implications for Effective.
Cancer Regulation. National Cancer Institute Monograph, Raven
Press, New York. :

Mann, T.: The Biochemistry of Semen and of the Male Reproductive
Tract, pp. 193-200. J. Wiley and Sons, Inc., New York, 1964.

Pereira, W. E., Summons, R. E., Reynolds, W. E., Rindfleisch, T. C.,
and Duffield, A. M., "The Quantitation of Beta Amino-isobutyric Acid
in Urine by Mass Fragmentography." Clin. Chim. Acta 49, 401 (1973).

Fink, K., Henderson, R. B., and Fink, R. M., Proc. Soc. Exp. Biol.
Med. 78, 135 (1951).

Young M. and Russell, W. T. “An Investigation into the Statistics of
Cancer in Trades and Professions." Spec. Rep. Soc. Med. Res. Com.
No. 99. London, 1926.

‘King, H., Bailar, J. C.: “Epidemiology of Urinary Blader Cancer."
J. Chron. Dis. 19, 735 (1966).