phase i dose de-escalation trial of a …...vol. 4, 303-310, february 1998 clinical cancer research...

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 March 19, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

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-



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



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).



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)


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



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


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




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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