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Vol. 4, 303-310, February 1998 Clinical Cancer Research 303
Phase I Dose De-escalation Trial of a-Difluoromethylornithine in
Patients with Grade 3 Cervical Intraepithelial Neoplasia’
Michele Follen Mitchell,2
Guillermo Tortolero-Luna, J. Jack Lee,
Walter N. Hittelman, Reuben Lotan,
J. Taylor Wharton, Waun K. Hong, and
Kenji Nishioka
Departments of Gynecologic Oncology [M. F. M., G. T-L., J. T. W.],
Biomathematics [J. J. L.], Clinical Investigation [W. N. H.], Tumor
Biology [R. LI, Thoracic/Head and Neck Medical Oncology[W. K. H.], and Surgical Oncology [K. N.], The University of TexasM. D. Anderson Cancer Center, Houston, Texas 77030
ABSTRACT
a-Difluoromethylornithine (DFMO) is a suicide inhib-
itor of ornithine decarboxylase and potent antiproliferative
chemopreventive agent. We conducted a dose de-escalation
Phase I trial of DFMO in patients with grade 3 cervicalintraepithelial neoplasia to determine an optimal dose of
DFMO using ornithine decarboxylase activity and poly-amine modulation as surrogate biomarkers and to evaluate
its toxicity. Thirty patients with biopsy-confirmed grade 3
cervical intraepithelial neoplasia were assigned sequentially
to one of five DFMO doses (1.000, 0.500, 0.250, 0.125, or
0.060 g/m2) given daily for 31 days. One patient was ex-
cluded from analysis for protocol violation. Polyamine levels
were assessed in cervical tissue, plasma, and RBCs. Tissue
and blood samples were obtained before and after treatmentwith DFMO. All patients underwent loop excision of thecervix at the end of the study for complete histological
evaluation and definitive treatment of the premalignant con-dition. No major clinical toxicity was observed at any DEMO
dose. A reduction in tissue spermidine to spermine (SPD:
5PM) ratio and an increase in plasma arginine levels wereobserved among patients receiving 1.000 g/m2/day (P <
0.05). A nonsignificant reduction in SPD:SPM ratio was also
observed in the 0.500 g/m2/day dose group, and a nonsignif-icant increase in plasma arginine level was observed down tothe 0.125 g/m2/day dose level. There was no evidence ofmodulation of other polyamines or precursors. Fifteen pa-tients experienced a complete (5 patients) or partial (10
Received 3/18/97; revised 10/21/97; accepted 11/10/97.The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to
indicatethisfact.1 Supported by National Cancer Institute Contracts N0l-CN-25433Aand N0l-CN-25433B.2 To whom requests for reprints should be addressed, at the Departmentof Gynecologic Oncology, Box 67. The University of Texas M. D.Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX77030. Phone: (713) 792-2770; Fax: (713) 792-7586.
patients) histological response. In conclusion, DEMO waswell tolerated and significantly modulated tissue SPD:SPM
ratio and plasma arginine level at the dose of 1.000 gIm2/day.To clarify whether DEMO has activity at lower doses, these
results will be tested in a three-armed double-blinded Phase
II study using placebo and DEMO doses of 0.500 and 0.125g/m2/day.
INTRODUCTION
CIN3 remains an important health problem for women,
particularly socially disadvantaged women, in both developed
and developing countries (1). An estimated 450,000 women
worldwide are diagnosed with cervical cancer annually (2). In
the United States in 1996, approximately 15,700 new cases of
invasive cervical cancer and 65,000 cases of carcinoma in situ
were diagnosed, and 4,900 women were expected to die from
this disease (3). High success rates and low complication rates
have been reported using ablative and excisional surgical pro-
cedures for the treatment of CIN. However, chemoprevention
represents a new, less invasive and potentially less expensive
alternative for the prevention of cervical cancer (4, 5). The use
of chemopreventive agents for the prevention of invasive cer-
vical cancer is also attractive for the following reasons: (a) the
cervix is an accessible organ for close follow-up examinations
by colposcopy, cervicography, Pap smear, and biopsy; (b) CIN
is considered a prototype for the study of intraepithelial neopla-
sias with a long premalignant stage; (c) CIN 3 is considered a
suitable intermediate end point for chemoprevention; and (d) the
theory of field carcinogenesis suggests that a systemic approach
rather than a surgical approach should be a more effective
therapy (5, 6). In addition, chemoprevention might be an ideal
therapeutic approach in developing countries, where access to
colposcopy and ablative or excisional treatment for CIN is
scarce.
Polyamines (putrescine, SPD, and SPM) are organic poly-
cations known to play important roles in many biological func-
tions (7). Polyamines are involved in maintenance, proliferation,
differentiation, and transformation of mammalian cells (8, 9). In
addition, polyamines have been shown to be involved in apop-
tosis and angiogenesis (10-12). ODC is a key enzyme in poly-
amine biosynthesis. ODC catalyzes the conversion of ornithine
to putrescine, a critical step in the biosynthesis of polyamines.
The gene encoding ODC is considered a putative proto-onco-
gene that is crucial for the regulation of cellular growth and
transformation (7, 9). Specific inhibition of ODC is one of the
3 The abbreviations used are: CIN, cervical intraepithelial neoplasia:CIN 3, grade 3 CIN; ODC, ornithine decarboxylase: DFMO, a-ditlu-oromethylornithine: HPV, human papillomavirus: SEB, surrogate end
point biomarker; SPD, spermidine: 5PM. spermine.
Research. on August 7, 2019. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
304 DFMO in Cervical Intraepithelial Neoplasia
most effective mechanisms for inhibiting polyamine biosynthe-
sis (7).
DFMO is a specific suicide inhibitor of ODC that has
shown antitumor and antimetastasis activity in several carcino-
gen-induced animal models, including colon, bladder, trachea,
breast, stomach, liver, intestine, oral cavity, and skin carcino-
genesis (7-9, 12, 13). Inhibition of ODC results in a major
reduction in putrescine and SPD levels and a small reduction in
5PM level. On the basis of this animal research, clinical studies
of DFMO in several high-risk populations have been conducted
to determine the minimal effective dose, to evaluate toxicity,
and to identify potential SEBs for monitoring carcinogenesis in
chemoprevention trials (10, 12-18). DFMO has been found to
produce few toxic effects in these animal and clinical studies (7,
13-16, 19). The two major adverse effects reported in clinical
studies are thrombocytopenia and hearing loss, which reversed
after discontinuation of treatment. These side effects are re-
ported particularly when DFMO is used at high doses as a
chemotherapy agent ( 19). Other, minor side effects include
anemia, leucopenia, diarrhea, fatigue, joint pain, insomnia, and
rash.
The potential of DFMO as a chemopreventive agent for
cervical cancer has not been assessed. We conducted a Phase I
dose dc-escalation trial of DFMO among women with a histo-
logical diagnosis of CIN 3 and evaluated the modulation of
ODC activity and levels of polyamines and their precursors in
tissue, plasma, and RBCs as SEBs of biological response. The
objectives of the study were: (a) to determine the optimal dose
and the minimum effective dose of oral DFMO for modulating
synthesis of polyamines and their precursors; (b) to assess the
toxicity oforal DFMO given in daily doses; and (c) to assess the
role of ODC activity and polyamine values in tissue and RBCs
and ornithine and arginine levels in plasma as SEBs of biolog-
ical response.
PATIENTS AND METHODS
Patient Eligibility. Patients were identified among
women attending the Colposcopy Clinic of the University of
Texas M. D. Anderson Cancer Center, Department of Gyneco-
logic Oncology. Patients were eligible if they met the following
criteria: they were not pregnant, they were 1 8 years or older, and
they had a histologically confirmed diagnosis of CIN 3, a lesion
involving at least one-third of the surface of the cervix, no prior
history of malignancy, a Zubrod performance status of �2, a
granulocyte count of >1500, a platelet count of >100,000,
serum bilirubin and creatinine levels of �l.5 mg/l00 ml, a
fasting triglyceride level less than twice the normal value, and
normal audiometry test results. In addition, premenopausal
women were required to have an adequate form of contraception
(such as hormonal contraception or bilateral tubal ligation) and
could not have childbearing plans for the length of this study.
Between March 10, 1994, and March 1, 1995, 143 patients
with a referral diagnosis of CIN 3 were screened for eligibility.
Seventy-five (52%) were ineligible because of small lesion size
or a histological diagnosis other than CIN 3. Of the 68 eligible
patients, 30 (44%) agreed to participate in the study. Reasons for
not participating included lack of transportation, unwillingness
to make additional clinic visits, or unwillingness to delay de-
finitive therapy.
Baseline Evaluation. Before enrollment, all participants
gave a complete medical history and underwent a physical and
pelvic examination; a Pap smear; gonorrhea and chlamydia
cultures; HPV testing; colposcopic examination of the vulva,
vagina, and cervix; and a risk factors and dietary assessment. In
addition, blood samples were collected for complete blood
count; serum electrolyte, chemistry, and coagulation studies;
serum levels of luteinizing hormone, follicle-stimulating hor-
mone, progesterone, and estradiol; plasma levels of DFMO,
ornithine, and arginine; and RBC levels of putrescine, SPD, and
5PM. Colposcopically directed biopsies from normal and ab-
normal areas were taken for permanent sections and for deter-
mination of ODC, putrescine, SPD, and 5PM levels. HPV
testing was performed by dot blot hybridization (ViraPap/Vira-
Type, Digene Diagnostics, Washington, DC), and negative
specimens were subjected to PCR analysis. Baseline audiograms
were conducted in all women before enrollment. This protocol
was reviewed and approved by the institutional review board,
and informed consent was obtained from each patient.
Study Protocol and Posttreatment Evaluation. Six pa-
tients were assigned sequentially to each of five doses of
DFMO: 1.000, 0.500, 0.250, 0.125, and 0.060 g/m2/day for 31
days. DFMO was provided by the National Cancer Institute in
elixir form in 200 mg/ml vials. Patients were provided with a
5-m13 syringe to ensure precise dosage. All patients were called
weekly by a research nurse, who recorded any toxic effects and
adherence problems and reported them to the principal investi-
gator (M. F. M.). All participants were instructed to keep a diary
of potential toxic effects. Clinical data were entered weekly by
a research nurse into the institutional patient data management
system. Patients were required to return their medication bottles
at the end of the study for quantification of total amount used.
Posttreatment evaluation included an audiogram, colpo-
scopic examination, and colposcopically directed biopsies from
normal and abnormal areas of the cervix for analysis of ODC
activity and polyamine (putrescine, SPD, and 5PM) levels. In
addition, blood samples were collected for determination of
posttreatment plasma levels of DFMO, ornithine, and arginine
and RBC levels of putrescine, SPD, and 5PM. After biopsy
samples from colposcopically normal and abnormal areas were
obtained, all patients underwent a cervical loop electrosurgical
excisional procedure for complete histological assessment and
definitive treatment of the premalignant condition. All histolog-
ical specimens were evaluated by three independent pathologists
who were blinded to the DFMO dose group of each patient.
Response was evaluated by the degree of histological regression
of the lesion; a complete response was defined as the absence of
a cervical lesion (negative histology) following DFMO treat-
ment, and a partial response was defined as a downstaging of
CIN status to CIN 1 or CIN 2.
Polyamine Analysis. Polyamine analysis of tissue sam-
pies (biopsy specimens) and blood was performed by one of the
authors (K. N.). Biopsy specimens and blood samples, after
separation into RBC and plasma fractions by centrifugation,
were frozen at -70#{176}C until analyses were performed. Each
sample was analyzed in duplicate, and pre- and posttreatment
samples were analyzed simultaneously to reduce technical var-
Research. on August 7, 2019. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Table I Patient characteristics (n 29) values were assessed by the difference between pre- and post-
treatment plasma values for each DFMO dose group. In addi-
tion, the proportional change and its 95% confidence intervals in
polyam�� values in tissue and RBCs and in ornithine and
Dose Age HPV status Days of
Patient no. (g/m2/day) (yr) (ViraPap) Lesion size” treatment
I 1.000 24 Positive >2ti 322 1.000 41 Positive <Y3 32 arginine values in plasma were estimated as the changes due to
3 1.000 30 Not tested V3_2/.1 29
� � � �:�: �2 �30 1 .000 35 Positive < V� 3 1
DFMO treatment (polyamine valuef�(,S�DFMO - polyamine val-
uebaseline)/polyamine valuebaselifle. Because of the large variabil-
ity in the measured values of polyamines and their precursors,
4 0.500 27 Negative #{189}_2/3 29 data analysis was performed by the use of nonparametric tests.
5 0.500 40 Negative < #{189} 256 0.500 28 Negative < #{189} 31
25 0.500 37 Positive <#{189} 31
26 0.500 20 Positive < #{188} 3 1
Differences in age distribution between DFMO dose groups
were assessed by the Kruskal-Wallis test. The association be-. .
tween age and baseline values of polyamines and thelr precur-
7 0.250 25 Negative #{189}-�/� 31 sors and the effect of elapsed time between last DFMO dose and
8 0.250 23 Positive < #{189} 31 posttreatment plasma DFMO levels were evaluated by the9 0.250 27 Negative #{189}��2ti 32
22 0.250 29 Negative < #{189} 3 1
23 0.250 29 Positive #{189}_2/i 26
5pe�nan’s rank correlation coefficient. The effect of HPV. . . .
status (positive/negative) on baseline values of polyamines and
24 0.250 25 Positive < #{189} 31 their precursors was assessed by the Mann-Whitney test. The
10 0.125 39 Positive I/3.2Z1 32 effect of DFMO treatment was assessed by comparing the1 1 0.125 25 Positive ‘/3 29
12 0.125 31 Negative I/t...2/3 2219 0.125 26 Positive #{189}_2/3 2720 0.125 22 Negative <#{189} 33
21 0.125 40 Positive #{189}_2ti 31
baseline and posttreatment values of polyamines and their pre-
cursors using the Wilcoxon matched-pairs signed-rank test. Sta-. . . .
tistical signtficance was set at an a of < 0.05 based on a
two-sided test.
I 3 0.060 22 Negative < ti 31
14 0.060 26 Positive >2/i 30
15 0.060 22 Negative <#{189} 31 RESULTS16 0.060 23 Positive #{189}�2A 29
17 0.060 36 Negative <#{189} 22
18 0.060 40 Negative < #{189} 31
. . . . . .
Thirty patients (six per dose group) met the eligibility
criteria and agreed to participate in the trial. However, one
patient enrolled in the 0.500 g/m2/day DFMO dose group took.
the wrong dose (estimated dose 0.080 g/m/day) and was ex-
cluded from this analysis. The median age of the 29 evaluable
patients was 27 years (range, 20-41 years). Seventy % (20 of
29) were white, 23% (7 of 29) were Hispanic, and 7% (2 of 29)
‘ . .
Proportion of the surface of the cervix affected by CIN (before
treatment).
iation. A 25% tissue homogenate was prepared using a Polytron were African-American. These characteristics were consistent
homogenizer (Brinkman Instruments, Westbury, NY) as de- with those of our colposcopy clinic population. There were no
scribed previously (15). A portion of the homogenate (20 p.1) differences in age by DFMO dose group (P 0.87). In 59%
was mixed with 80 �il of 5% sulfosalicylic acid, sonicated, and (17) of the 29 women, cervical lesions were one-third or less the
microcentrifuged (13,000 X g) for 15 mm at room temperature size of the surface of the cervix, in 34% (10 of 29), the lesions
to obtain a clear supernatant for polyamine analysis. The re- were between one-third and two-thirds the size of the surface of
maining portion of the homogenate was centrifuged (700 X g) the cervix, and in 7% (2 of 29), the lesions were greater than
for 15 mm at 4#{176}C,and the supernatant was analyzed for ODC two-thirds the size of the surface of the cervix. Eighty-three %
activity and protein levels. Protein concentrations were deter- of the women were positive for HPV by dot blot hybridization
mined using Bio-Rad (Richmond, CA) protein assay kits. A new and PCR. A detailed description of patient characteristics is
procedure, using O-phthalaldehyde in a Dionex BioLC high- presented in Table 1.
performance liquid chromatography unit equipped with an Overall, patients were compliant with DFMO intake and
HPLC-CS2 column and postcolumn detection system (Dionex, their follow-up visits. The mean number of treatment days was
Inc., Sunnyvale, CA), was used for determinations of free poly- 30 days (median 31 days, range 22 to 33 days). Four patients
amine levels. Arginine, ornithine, and DFMO levels were meas- took DFMO for 32 days and one for 33 days. Patients (I 1 of 29)
ured by the method described by Grove et al. (20). Plasma who reported fewer than 3 1 days of DFMO treatment missed an
DFMO levels are reported in pmol/ml, plasma arginine and average of 4 days of treatment (median, 4 days; range, 1-9
ornithine levels in nmol/ml, tissue polyamine levels in pmol/mg days). Posttreatment plasma DFMO levels are shown in Table 2.
of soluble protein, and RBC polyamine levels in nmol/ml of A large, but not statistically significant, difference in median
packed RBCs. plasma DFMO values between patients in the highest (73.9
Statistical Analysis. Posttreatment DFMO values in pmol/ml) and lowest (5.4 pmol/ml) dose groups was detected
plasma were compared by dose group. The effects of DFMO (Kruskal-Wallis test, P 0.12). Plasma DFMO values ranged
treatment on ODC, polyamine (putrescine, SPD, and SPM) from below detectable in the 0. 125 g/m2/day dose group to a
values, and SPD:SPM ratio were assessed for each DFMO dose high of 621.9 pmol/ml in the 1 .000 g/m2/day dose group.
group by the differences between pre- and posttreatment values Although women who took the highest doses had higher plasma
in biopsy specimens from abnormal areas of the cervix and in DFMO values than women who took the lowest doses, substan-
RBCs; similarly, the effects of DFMO on ornithine and arginine tial variability and overlapping in plasma DFMO values were
Clinical Cancer Research 305
Research. on August 7, 2019. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Table 2 Posttreatment plasma DFMO levels
DFMO DFMO level (pmollml)
Table 3 Adverse events probably related to DFMO treatment
C, One patient was excluded from analysis for protocol violation.
I, Below detectable value.
Dose
(g/m2/day)
1.000
0.500
0.250
0.125
0.060
Adverse event
DiarrheaDizzinessMalaise
Mood changes
Motor
Myalgia
NauseaNosebleed
Pruritus
Skin rash
Stomatitis
DiarrheaDizziness
Nausea
Skin rash
Stomatitis
Muscle crampsSkin rash
Anorexia
Conjunctivitis
DiarrheaFatigue
Indigestion
MoodNausea
Nosebleed
Skin rash
Stomatitis
Mood changesMuscularStomatitis
Grade
1-2
1-2
1-2
1-3
21-2
1-3
2
No. of
patients
(patient no.)
2 (1, 2)
1 (29)1 (1)1 (2)
I (2)
1 (1)
3 (2, 29, 30)
2 (1, 2)
1 (1)
1 (2)3 (1. 2, 30)
I (4)1 (25)1 (4)1 (25)
I (5)
I (22)
I (7)
1 (12)I (12)
2(11, 19)
2(10, 19)
I (11)
1 (11)
3(11. 16. 19)
I (II)1 (11)3 (10, 1 1, 12)
1 (14)I (16)1 (16)
306 DFMO in Cervical lntraepithelial Neoplasia
(g/m2/day) patients Mean (±SE) Median Minimum Maximum
1.000 6 146.3 (96.4) 73.9 1.8 621.90.5(X) 5” 23.0 (8.2) 24.7 0.6 42.1
0.250 6 23.9 (4.6) 28.9 2.8 31.4
0.125 6 16.5 (5.3) 24.5 33.2
0.060 6 5.9 (0.7) 5.4 4.3 8.8
observed across dose groups. Furthermore, values were similar
for the intermediate dose groups (0. 125-0.500 g/m2/day).
The median elapsed time between the final DFMO dose
and the collection of posttreatment biological specimens was
3.5h. In 28 patients, specimens were collected within 24 h after
the last dose of DFMO. Furthermore, the majority of patients
(23 of 29) had specimens collected within S h after their last
DFMO dose (the other intervals were 14 h in 1 patient, 15 h in
2, 1 6 h in 2, and I 96 h in 1 ). Although lower plasma DFMO
levels were observed in five of the six women whose elapsed
times were >5 h (ranging from below detectable values in the
patient with the 196 h ofelapsed time to 8.4 pmollml in a patient
with an elapsed time of 15 h), there was no statistically signif-
icant correlation between the elapsed time after final DFMO
dose and posttreatment plasma DFMO levels (Spearman’s rank
correlation coefficient = -0.176; P = 0.362).
Toxicity. No major adverse effects were reported in any
of the DFMO dose groups. The most frequently reported ad-
verse effects, possibly related to DFMO treatment, included
stomatitis, nausea, diarrhea, and dizziness (Table 3). Similar
symptoms were reported at all doses, and most of these symp-
toms were classified as grade 1 . One patient, in the 0.125
g/m2/day dose group, developed severe (grade 3) stomatitis and
was required to stop the medication. The patient was examined
by one of the investigators (M. F. M.) 3 days after the onset of
stomatitis; several ulcerations were still present, but the patient
was tolerating food well, and her WBC and differential counts
were normal. A dental consultation and a culture from the
ulcerations for herpes virus were obtained. Although the culture
was negative, acyclovir administration resulted in relief of the
pain and disappearance of the lesions within 48 h. All other
patients with stomatitis only reported “mouth soreness”; none
had visible lesions. No DFMO-induced ototoxicity was apparent
in any dose group. Baseline and posttreatment audiograms were
normal in 28 of the 29 patients included in this analysis. In one
patient, the baseline audiogram showed a mild conductive defect
of no clinical significance, which remained unchanged after
DFMO treatment. No other toxic effects were evident by labo-
ratory testing. There were no differences between baseline and
posttreatment blood counts, serum chemistry results, coagula-
tion times, or hormone levels.
DEMO Effect on ODC Activity and Polyamine Values.Tables 4 and 5 show the median values of polyamines and their
precursors in tissue, plasma, and RBCs before and after DFMO
treatment. A statistically significant (P < 0.05) decrease in
tissue SPD:SPM ratio and a statistically significant increase in
plasma arginine level were observed among patients in the
highest DFMO dose group (I .000 g/m2/day). A decrease in
tissue SPD:SPM ratio of women receiving 0.500 g/m2/day of
DFMO and an increase in plasma arginine level of women
receiving DFMO doses as low as 0. 1 25 g/m2/day were also
detected, but these changes were not statistically significant.
Similar findings were observed by calculating the proportional
changes in these variables (Figs. I and 2). No modulation of
ODC activity, other polyamines, or precursor markers in tissue,
plasma, or RBCs was detected.
DEMO Effect on CIN Lesions. Although the assess-
ment of histological response was not one of the primary ob-
jectives of this Phase I trial, a partial or complete histological
response was detected in 52% (15) of the 29 evaluable patients
despite the short period of treatment. A complete response
occurred in S patients, and a partial response occurred in 10
patients (Table 6). However, most responses (10 of I 5) were
observed among women receiving DFMO doses � 0.250 g/m2/
day. Among women receiving the highest DFMO doses (0.500
and I .000 g/m2/day), only partial responses were detected.
Details of patient characteristics and assay results for re-
sponders are shown in Table 7. Response rate was not associated
with age, lesion size, HPV status (positive/negative), or modu-
lation of SPD:SPM ratio and arginine level (data not shown).
Research. on August 7, 2019. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1.000Baseline
Posttreatment
0.500
Baseline
Posttreatment
0.250BaselinePostttreatment
0.125
Baseline
Posttreatment
56 (29-73)
64 (30-1 10)
45 (35-61)
47 (43-50)
52 (35-76)
57 (39-71)
51 (27-63)
65 (3 1-85)
62 (51-87)
76 (42-95)
80(47-113)
92 (47-126)
73 (55-113)
81 (40-141)
1 1,332 (9,750-36,194)12,825 (8,993-25.928)
9,773 (6,377-24,514)
8,648 (3,669-17,428)
16,1 15 (6,581-32,611)12,608 (5.274-18.800)
25,491 (10,340-32,780)
21,391 (12,358-40,780)
10,612 (4,739-17,583)
9,33 1 (6,392-23,380)
126(109-232) 14,311 (7,399-27,724)108 (74-305) 15,823 (10,601-20,645)
Clinical Cancer Research 307
Table 4 Comparison of pre- and posttreatment polyamine median values in abno rmal tissue by DFMO dose level (ii 29)
DFMO dose level Tissue ODC, Tissue putrescine, Tissue SPD, Tissue 5PM, Tissue SPD:SPM ratio
(g/m2/day) median (range) median (range) median (range) median (range) (range)
1.000Baseline 168 (66-675) 640 (496-1586) 3993 (1,270-15,400) 3,951 (1,194-16,831) 1.06(0.91-1.21)”
Posttreatment 190 (75-627) 885 (255-4,943) 6,562 (1,628-14,655) 6,839 ( 1,924-21,832) 0.90 (0.53-4)97)
0.500
Baseline 182 (103-294) 490 (68-1,925) 2,046 (812-1 1,023) 2,199 (846-9.229)” 1.00 (0.93-1.19)
Posttreatment 63 (23-395) 243 (154-1532) 3,267 (1,601-12,564) 3.763 ( 1,663-16,050) 0.87 (0.6l-().97)
0.250Baseline 155 (81-1,812) 1,161 (464-2,019) 4,1 15 (2,166-5,873) 4,226 (2,542-7,997) 0.88 (0.64-1.19)
Posttreatment 204 (98-327) 822 (599-2,458) 3,832 ( 1,972-20,537) 3,986 ( 1,847-13,070) 1 .07 (0.72-1.57)
0.125
Baseline 327 (106-660) 1,580 (590-1,867) 3,728 (1,932-7,483) 3,777 (2,139-1 1,754) 0.87 (0.64-1.46)
Posttreatment 236 (40-1,534) 724 (1 13-1,1 19) 2,854 (2,120-9,895) 3,019 (1,477-9,498) 0.99 (0.83-1.44)
0.060
Baseline 331 (134-598) 1,465 (1,056-1,924) 7,044 (2,895-10,513) 6,284 (1,410-14,809) 0.83 (0.55-7.46)
Posttreatmenta p < 0.05 by t
659 (634-1,054) 579 (130-2,276) 6.413 (3,232-1 1,219)
he Wilcoxon matched-pairs signed-ranks test.
5,718 (2,273-l5,992L 1.14 (0.50-1.49) �
Table 5 Comparison of pre- and posttreatment polyamine median values in plasma and RBCs by DFMO dose level (ii = 29)
DFMO dose level Plasma ornithine, Plasma arginine, RBC putrescine, RBC SPD, RBC SPM.
(g/m2/day) median (range) median (range) median (range) median (range) median(range)
0.060Baseline 40 (20-67) 83 (73-100)Posttreatment 40 (25-52) 67 (55-128)
a p < 0.05 by the Wilcoxon matched-pairs signed-ranks test.
78 (47-1 15)” 40 (26-90) 15,660 (10,877-19,3 13)
106 (5-144) 48 (0-91) 17,084 (14,489-2 1,468)
99 (34-l8ly’ 17,81 1 (12,481-24,924)
75 (0-88) 15,980 (14,022-25,436)
91 (63-205) 16,229(11,076-25,575)
88 (0-196) 12,383 (7,629-19,926)
73 (29-135) 12,132 (6,931-15,003)
89(0-369) 16,271 (2,637-22,179)
These observations were confirmed by computer-assisted quan-
titative analysis of Feulgen-stained slides.
DISCUSSION
No major side effects were associated with DFMO intake
in this Phase I dose de-escalation trial for patients with a
histological diagnosis of CIN 3. Results from this trial show that
DFMO is well tolerated in doses of 1.000 g/m2/day or lower
given for a short period of time (31 days). Particularly important
was the absence of ototoxic effects at these doses. The side
effects reported in our study are similar to those described by
Love et a!. (14), Creaven et al. (15), Meyskens et a!. (16), and
Loprinzi et a!. ( 19). The most common ones experienced in our
study were nausea, diarrhea, dizziness, and mouth soreness or
stomatitis. Most of these symptoms were classified as grade 1
toxic effects and were observed at all DFMO dose levels.
Although in most patients (23 of 29), plasma DFMO values
were measured within S h after their last DFMO dose, our data
did not show a direct relationship between dose and plasma
level. We were unable to explain the extremely high plasma
DFMO level observed in a patient receiving 1 .000 g/m2/day
(621.9 pmol/ml). Reanalysis of this sample provided similar
values, and the elimination of this case from the statistical
analysis did not modify our results. The lower plasma DFMO
values observed in most of the patients with longer elapsed
times are consistent with prior studies that have shown a short
half-life of DFMO in plasma (12, 18). After oral or iv. admin-
istration, DFMO reaches a peak plasma concentration within 6 h
and has a mean half-life of 3.5-5.6 h ( I 2, 18).
Several groups have written extensively about polyamine
measurement issues in other organs, particularly the colon (17).
Previous studies have shown a large intra- and interindividual
variability in polyamine values. Despite this variability, poly-
amine synthesis has been shown by several investigators to be
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0
0.C,,
a0.C,,C
C,C)CIC
C-,
ICC0
.tI
C,CC
El
C
C,C)CIC
.0C)
ICC0
t8.2
0.CICC,
1.000 0.500 0.250 0.125 0.060
DFMO dose g/m’Iday
1.000 0.500 0.250 0.125 0.060
308 DFMO in Cervical Intraepithelial Neoplasia
Fig. I Proportional changes (squares) and 95% confidence intervals
(error bars) in SPD:SPM ratio in abnormal tissue [(polyamine
value �,�,t)FMO polyamine value ,��,.1j,p)/polyamine value ba�eine]
Table 6 Num ber of histologic al responses by DFMO d ose level
Dose
(g/m�/day)
Response
Complete Partial None Total
1.0(X) 0 3 3 60.500 0 2 3 5
0.250 1 3 2 6
0.125 2 2 2 6
0.060 2 0 4 6
Total 5 10 14 29
modulated by DFMO and to be a potential SEB of cellular
proliferation. In a preliminary study, our group had higher ODC
activity in tissue biopsy specimens from colposcopically abnor-
mal areas of the cervix than in those from colposcopically
normal tissue (21). The large variability in polyamine levels in
cervical tissue observed in this study, both before and after
DFMO treatment, is consistent with results from other organ
sites (14-18, 22).
Hixson et a!. ( 17) observed statistically significant higher
values of putrescine, SPD, and 5PM in large biopsy specimens
than in smaller biopsy specimens in patients undergoing
colonoscopy, but no differences in ODC activity and SPD:SPM
ratio were detected. The site of the biopsy specimen, the use of
individual biopsy or the pooled lysate of multiple biopsies (four
samples), and the date of laboratory assay were found to be
sources of variation in the measurement of ODC activity and
polyamine values in colorectal tissue. Wang et a!. (22), in a
small case-control study, demonstrated that among patients at
high risk for colorectal cancer, a larger number of biopsies per
2.0
1.5
�‘ T1 �1T
DFMO dose g!m’/day
Fig. 2 Proportional changes (squares) and 95% confidence intervals
(error bars) in plasma arginine levels [(polyamine value �
polyamine value baseIine)/P#{176}lYam�� value baseline]’
patient (4-8 biopsies versus 1-4 biopsies) provides a more
reliable measurement of polyamines in colorectal tissue. How-
ever, the size of the uterine cervix might be an inherent limita-
tion for the collection of multiple biopsies. Furthermore, it has
been suggested that obtaining multiple cervical biopsies might
have an effect on the natural history of the disease.
Bacterial contamination has also been reported to have an
impact on ODC activity and polyamine content and therefore
may mask any potential effect of DFMO. Boyle et a!. (23)
observed that changes in ODC activity and polyamine content in
exfoliated buccal mucosal cells were due primarily to bacterial
contamination, and the effect of oral DEMO therapy was not
accurately determined. However, no effect of bacterial contam-
ination was observed in biopsy specimens from the rectal mu-
cosa. In our study, in an attempt to reduce the level of variability
in polyamine measurements, all biopsy specimens were rinsed
with saline solution before freezing to reduce bacterial contam-
ination, and all specimens were analyzed in a single batch and
run in duplicate to reduce laboratory error (21).
Statistically significant modulation of polyamines and their
precursors by DFMO was only observed for the tissue SPD:
5PM ratio and plasma arginine level among patients receiving
1 .000 g/m2/day. Nonsignificant modulation of SPD:SPM ratio
was observed among women receiving 0.500 g/m2/day and of
arginine level among women receiving doses as low as 0.125
g/m2/day. No other effects on polyamine biosynthesis were
detected after DFMO treatment. Our data did not demonstrate an
increase in ornithine level in plasma; a decrease in putrescine,
SPD, and 5PM levels in RBCs and cervical tissue; or a decrease
in ODC activity in cervical tissue. On the basis of these results,
it is difficult to determine the minimum effective dose of DFMO
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Clinical Cancer Research 309
Table 7 Descriptio n of responders
DFMOProportio nal change
Patient Dose Age Treatment Time since plasma level SPD:SPM
no. (g/m2/day) (yr) HPV status Lesion size Response days dose (hr)” (pmol/ml) Arginine ratio
1 1.000 24 Positive >2/i Partial 32 1.0 59.5 -0.04 1.27
2 1.000 41 Positive <#{189} Partial 32 2.0 12.6 0.28 -0.05
3 1.000 30 Positive /i_2/s Partial 29 16.0 1.8 0.59 0.144 0.500 27 Negative #{189}��2Z� Partial 29 14.0 0.6 0.09 1.9825 0.500 37 Positive <#{189} Partial 31 3.0 42.1 0.39 0.32
7 0.250 25 Negative #{189}-�/� Partial 31 15.0 29.7 -0.59 -0.52
8 0.250 23 Positive <#{189} Partial 31 16.0 2.8 1.1 1 -0.59
9 0.250 27 Negative I/i.2/i Partial 32 3.0 20.2 0.02 2.48
22 0.250 29 Negative <#{189} Complete 31 4.0 31.4 -0.02 -0.10
1 1 0.125 25 Positive <‘/3 Complete 29 1.5 4.2 0.08 1.63
19 0.125 26 Positive I/3..2/1 Complete 27 2.5 33.2 0.31 -0.0720 0.125 22 Negative <‘/i Partial 33 3.0 24.5 0.03 0.01
21 0.125 40 Positive A�2/� Partial 31 4.0 26.3 0.28 -0.08
13 0.060 22 Negative <#{189} Complete 31 3.0 5.6 0.10 0.06
18 0.060 40 Negative <#{189} Complete 31 2.5 4.3 -0.19 -0.45
C’ Elapsed time between the final DFMO dose and the collection of posttreatment biological specimens.
that would suppress polyamine biosynthesis and to establish the
role of polyamines as SEBs of biological response in cervical
carcinogenesis.
Other authors have also been unable to determine the
minimum effective dose of DFMO in other tissues. On the basis
of toxicity, Love et a!. (14), in a Phase I study, determined that
0.500 g/m2/day was an adequate dose for future evaluation.
Creaven et a!. (15) and Pendyala et a!. (18) observed modulation
of putrescine and SPD values in urine at a dose level as low as
0.200 g/m2/day. Meyskens et a!. (16) demonstrated modulation
of the SPD:SPM ratio in colorectal mucosa at a dose of 0.100
g/m2/day and suggested that a dose of 0.050 g/m2/day should be
assessed.
Although the response rate observed in this study is en-
couraging and consistent with the biological mechanism of
DFMO and its effect on polyamine biosynthesis, these data need
to be interpreted with caution because of the scope and limita-
tions of the study design, the lack of a control group, and the
lack of association between DFMO dose level and response rate
and type of histological response (complete or partial).
In conclusion, this study supports the low toxicity of
DFMO as a chemopreventive agent when used at these doses for
a short period of time. It confirms the large interindividual
variability of polyamine values that has been observed in other
human tissues by other authors. It also suggests that SPD:SPM
ratio and arginine value can be modulated by DFMO. Our data
suggest that short-term administration of DFMO may be capable
of inducing regression of CIN lesions, but these findings need
further evaluation in a randomized, double-blinded Phase II
trial. In addition, this study suggests that low-income women
attending our colposcopy clinic can be successfully recruited,
treated, and followed in short-term chemoprevention trials.
However, we were unable to determine in this study the mini-
mum effective DFMO dose for suppressing polyamine biosyn-
thesis in cervical tissue or to establish the role of polyamines as
potential markers of biological response in cervical neoplasias.
To better address the efficacy of DFMO in this high-risk pop-
ulation (women with CIN 3), a three-armed double-blinded
Phase II clinical trial is planned using a placebo and DFMO
doses of 0.500 and 0. 125 g/m2/day. Given that there was no
association between dose level and histological response in this
study, our rationale in selecting these two dose levels for a
future Phase II trial is driven by the nonsignificant modulation
of SPD:SPM ratio and arginine level observed at doses lower
than I .000 g/m2/day and by the need to further assess, in a larger
population, the effect of intermediate doses on histological
response and biological markers, as well as the value of these
markers for evaluating chemoprevention with DFMO.
ACKNOWLEDGMENTS
We acknowledge Drs. Elvio Silva, Anais Malpica, and bun Boiko
for the reading of all histological material; Rosanna Lyon, research
nurse, for conducting the study; Sunita Patterson for editing; and Pat
Williams and Ronetta Fairchild for preparation of the manuscript.
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1998;4:303-310. Clin Cancer Res M F Mitchell, G Tortolero-Luna, J J Lee, et al. in patients with grade 3 cervical intraepithelial neoplasia.Phase I dose de-escalation trial of alpha-difluoromethylornithine
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