decreases in levels of serum fibronectin predict the ...weight gain/edema minimal ankle pitting...

Vol. 2, 1705-1712, October 1996 Clinical Cancer Research 1705

Decreases in Levels of Serum Fibronectin Predict the Severity of

Vascular Leak Syndrome in Patients Treated with Ricin A

Chain-containing Immunotoxins’

Roxana Baluna, Edward A. Sausville,

Marvin J. Stone, Mary Alice Stetler-Stevenson,

Jonathan W. Uhr, and Ellen S. Vitetta2

Cancer Immunobiology Center and Department of Microbiology, TheUniversity of Texas Southwestern Medical Center at Dallas, Dallas,

Texas 75235-8576 ER. B., J. W. U., E. S. V.]; DevelopmentalTherapeutics Program, Division of Cancer Treatment, Diagnosis, and

Centers [E. A. S.] and Laboratory of Pathology, Division of ClinicalScience [M. A. S-S.], National Cancer Institute, Bethesda, Maryland20892; and Baylor University Medical Center and Charles A.

Sammons Cancer Center, Dallas, Texas 75246 [M. J. S.]

ABSTRACT

The major dose-limiting adverse effect of ricin A chain-containing immunotoxin (IT) therapy is vascular leak syn-drome (VLS). Since plasma fibronectin (Fn) plays a role in

maintaining microcirculatory integrity and since the gradi-

ent between plasma and tissue Fn can be altered in variouspathological situations, we determined whether the admin-istration of IT-ricin A chain to patients resulted in changes

in the levels of serum Fn and, if so, whether these changes

correlated with the severity of VLS. We also measured theserum levels of tumor necrosis factor a (TNFa), a prom-

fiammatory cytokine which has been implicated in tissuedamage and in interleukin 2-mediated VLS. Our resultsindicate that the most severe manifestations of VLS wereassociated with the highest pretreatment levels of Fn, the

largest decreases in Fn immediately after starting IT ther-

apy, increases in the levels of serum TNFa, higher concen-

trations of circulating IT, and the lowest numbers of circu-

bating tumor cells. These parameters should, therefore, be

useful for predicting which patients will have severe VLS.

INTRODUCTION

The major dose-limiting adverse effect of IT� therapy in

humans is VLS. VLS is characterized by an increase in vascular

permeability accompanied by extravasation of fluids and pro-

teins from the capillary vessels into the tissues, resulting in

interstitial edema, a decrease in microcircubatory perfusion, and,

in its most severe form, multiple organ failure. VLS occurs in

Received 4/10/96; revised 6/24/96; accepted 6/25/96.I Supported by NIH Grants CA-28149 and CA-4l081 and a grant from

The Meadows Foundation.2 To whom requests for reprints should be addressed. Phone: (214)

648-1201; Fax: (214) 648-1205.3 The abbreviations used are: IT, immunotoxin; VLS, vascular leaksyndrome; RTA, ricin a chain; EC, endothelial cell; dg, deglycosylated;Fn, fibronectin; BI, bolus infusion; CI, continuous infusion; TNF-a,tumor necrosis factor a; IL-2, interleukin 2.

patients treated with ITs containing RTA, blocked ricin, saporin,

pokeweed antiviral protein, Pseudomonas exotoxin A, and diph-

theria toxin (1-7). VLS is not unique to II therapy and is a toxic

side effect of therapy with cytokines, growth factors, antibodies,

and of chemotherapy (8-16). VLS can also occur in a variety of

different diseases (17-20). The mechanisms underlying VLS are

not well understood, although in the case of II therapy, it has

been suggested that RTA alters EC functions required for vas-

cular integrity (21). In this regard, ECs are 10-fold more sensi-

tive to the in vitro nontargeted cytotoxic activity of dgRTA than

most other cell lines (21). In addition, dgRIA binds to Fn in

vitro, perhaps preventing it from binding to its receptors on ECs

and promoting disruptions of EC monobayers (2lA).

Fn is a multifunctional protein which exists in both a

soluble form in the plasma and other body fluids and in an

insoluble form in basement membranes, extracellular matri-

ces, and connective tissues (22). Plasma Fn plays a robe in

microcirculatory integrity since it is incorporated into the

extracellular matrix. It also augments the macrophage-medi-

ated clearance of circulating microaggregates (23-28). The

gradient between plasma and tissue Fn can be altered in

various diseases, particularly as levels of plasma Fn decrease.

One potential consequence is a deficiency in Fn-EC interac-

tions, resulting in an increase in vascular permeability. Once

the permeability of the vasculature increases, proteins and

cytokines might enter the tissues, where further exacerbation

of VLS may occur.

In this study, we determined whether the administration of

II-dgRTA to patients resulted in decreased levels of serum Fn

and, if so, whether these decreases correlated with more severe

VLS. We also correlated changes in Fn levels with the presence

of circulating tumor cells and concentrations of II in the same

sera.

MATERIALS AND METHODS

Patients. Sera obtained from patients with histologically

confirmed non-Hodgkin’s bymphoma of low, intermediate, or

high grade and previously treated with RIA-based ITs were

entered into these studies. The patients are described in detail

elsewhere (2, 29-31).

IT Preparation and Administration. The three ITs(Fab’-RFB4-RTA, IgG-HD37-SMPT-RTA, and IgG-RFB4-

SMPI-RIA) were prepared as described previously (2, 29). The

ITs were administered by either bolus infusion (BI) or contin-

uous infusion (CI). The CI was administered over 8 days at three

dose levels. BI included 4-h infusions given every other day

over a total of 8 days.

Assessment of Toxicity. The patient’s physical status,

hematology, blood chemistry, and urinalysis were assessed daily

during treatment, and all adverse effects were recorded and

graded from grades I to IV using criteria described previously

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1706 Serum Fn during Immunotoxin Therapy

Table 1 Grading of immunotoxin-related toxicities

Symptoms

Graded toxicity scale

Grade I Grade II Grade III Grade IV

Weight gain/edema Minimal ankle pittingedema

Ankle pitting edema Peripheral edema, weightand weight gain gain >10 lb, pleural

<10 lb effusion with nopulmonary deficit

Anasarca, severe pleuraleffusion with pulmonary

function deficit, ascites,pulmonary edema

Hypoalbuminemia >2.8 2.4-2.7 2.0-2.3 �2.0

(serum albumin

in g/dl)

Renal

Serum creatinine 1.25-1.5 X normal 1.5-3.0 X normal 3.1-6.0 X normal (4-7.9) >6.0 X normal; requiredincrease (mg/dl) (1.6-2.0) (2.1-3.9) dialysis (�8.0)

Irreversible loss of >20%

Creatinine clearance 70-80% of baseline 50-69% of baseline 30-49% of baseline <30% of baseline

PulmonaryPulmonary function FVC� 70-80% of predicted FVC 50-69% of predicted FVC <50% of predicted Unable to perform test due

abnormality FEV1 or DLCO 60-80%of predicted; 15-25%

decrease from abnormal

baseline

FEV1 or DLCO 40-59% FEV1 or DLCO <40% of

of predicted; 26-50% predicted; 50% decrease

decrease from abnormal from abnormal baseline

baseline

to respiratory distress

Respiratory symptoms Mild or transient,asymptomatic with

pulmonary function tests

abnormal

Dyspnea on significant Symptoms during normalexertion activity, persistent

dyspnea

Severe symptoms at restnonresponsive to Rx

Chest X-ray <10% of lung fields showinfiltrate or effusion

<20% of lung fields show <50% of lung fields showninfiltrate or effusion infiltrate or effusion

>50% of lung fields show

infiltrate or effusion

CardiovascularCardiac function Asymptomatic, � ejection

fraction by <20% of

baseline

Asymptomatic, � ejection Mild CHF, responsivefraction by >20% of to Rx

baseline

Severe, refractory CHF

Pericardial effusion Asymptomatic, No Rx

required

Pericarditis, (rub, chest pain, Symptomatic; large effusion,

EKG changes) drainage required no

tamponade, resp todrainage

Large effusion, tamponade;

drainage urgently required

Hypotension 10-20% � systolic,no Rx required (includestransient orthostatichypotension)

21-30% � systolic, 31-40% � systolic,required fluids or required pressorsother Rx but not and hospitalization,hospitalization resolves within 48 h

>40% � systolic, requiredhospitalization,unresponsive to pressors,

requires >48 h to resolve

after stopping agent

a FVC, forced vital capacity; FEV1, forced expiratory volume in 1 s; DLCO, diffusion capacity of the lungs for CO,; Rx, treatment; CHF,

congestive heart failure; EKG, eletrocardiogram.

(Refs. 2 and 29; Table 1). Peripheral edema, weight gain, pleural

effusions, ascites, anasarca, renal, pulmonary, and cardiac func-

tion deficits were adverse events used in grading VLS. We

classified these grades as: (a) mild: Grade I peripheral edema

and administration of the complete course of IT; (b) moderate:

grades Il-Ill edema and weight gain, no pulmonary function

deficit, administration of the complete course of II with possi-

ble delays; and (c) severe: grade IV edema, anasarca, pulmonary

function deficit, cardiac function deficit, and cessation of IT

therapy.

Serum Fn Levels Levels of serum Fn were determined

using duplicate serum samples in a commercial radial immu-

nodiffusion assay. [The Binding Site (Birmingham, United

Kingdom)].

Serum Cytokine Levels. Levels of TNF-a were deter-

mined using a commercial ELISA kit purchased from Immuno-

tech (Marseille, France).

Serum II Concentrations Serum IT concentrations

were measured using a RIA as described previously (29).

Flow Cytometric Analysis. The presence or absence of

peripheral blood-circulating tumor cells was assessed according

to previously described methods (2).

Statistical Analysis. All values are expressed as

means ± SE. For statistical analysis of the data, Student’s t test

and the Pearson correlation were used. Correlations between

parameters were assessed by linear regression analysis. A two-

tailed P < 0.05 was considered to represent a significant dif-

ference.

RESULTS

Patients. Sera from 56 patients with non-Hodgkin’s lym-

phoma were entered into this study. Patients had been treated by

BI or CI with a total of 65 courses of IgG-HD37-RTA (30),

IgG-RFB4-RTA (2, 31), or Fab’-RFB4-RTA (29). Seventeen

patients had received CI and 9 had received BI of IgG-RFB4-

RTA, and 7 had received CI and 18 had received BI of IgG-

HD37-RTA. Five patients received BI of Fab’-RFB4-RTA.



Fig. 1 The frequency of VLS manifesta-tions in patients treated with ITs.

0 20 40 60 80 100

Percent of patients

Clinical Cancer Research 1707

albumin decrease

weight gain/edema

pulmonary

insufficiency

cardiovascular

insufficiency

renal

insufficiency

Table 2 Grades and manifestat ions of VLS”

IT

IgG-HD37-dgA IgG-RFB4-dgA Fab’-RFB4-dgA

Regimen BI CI BI Cl BI

Cumulative doses (mg/m2) (range) 2-24 9.6-19.2 23-48 9.6-28.8 25-100VLS manifestations

Weight gain, edema 7/1/2” 2/2/2 5/2/1 15/2/7 3/1/0

Hypoalbuminemia 3/7/2 4/2/0 0/6/3 19/6/1 2/1/0

Renal 5/0/0 3/1/0 0/2/3 11/3/1 1/0/0

Pulmonary 1/2/0 0/2/0 0/3/1 4/2/3 1/1/1

Cardiovascular 2/0/0 0/0/0 3/1/1 2/2/1 4/0/0

CC Maximum toxicity grades (represented as the ratio of patients with grade Ilgrade Illgrades III and IV)

I, Number of patients with grades 11111111 and IV toxicity.

VLS. The major clinical features of VLS in these pa-

tients are described in Fig. 1 and Table 2. VLS was mild in

28 courses, moderate in 25 courses, and severe in 12 courses.

In patients with severe toxicity, the administration of IT was

halted. The most severe manifestations of VLS were pubmo-

nary edema and hypotension (Table 3). As shown in Fig. 2,

patients who had mild VLS had bower levels of serum Fn

prior to treatment than patients who had severe VLS. Serum

Fn levels decreased in patients with moderate VLS (mean ±

SE, -29 ± 3%, P < 0.01) and severe VLS (mean ± SE,

-49 ± 4%, P < 0.001) and increased in patients with mild

VLS (mean ± SE, 22 ± 3%, P 0.07; Fig. 3A). The changes

in levels of serum Fn occurred early and were sustained until

the completion of therapy (Fig. 3B). Unlike Fn, serum

albumin bevels and weight gains showed fewer marked

changes in patients with severe VLS. Thus, albumin bevels

decreased by 21, 25, and 28% in mild, moderate, and severe

VLS, respectively, and weights increased by 2, 3.8, and

4.5%, respectively. Furthermore, reductions in the bevels of

serum albumin and weight gains were more pronounced bate

in the course of infusion, whereas changes in the Fn bevels

occurred early (data not shown). The most significant de-

creases in the levels of serum Fn were also apparent in

patients with severe toxicity whose infusions were inter-

rupted (Table 3). The decreases in the levels of serum Fn

were observed during the first day of IT therapy in 83% of the

patients with severe toxicity. Serious manifestations of VLS

such as pulmonary insufficiency or hypotension were pre-

ceded by a decrease in the levels of serum Fn but not

necessarily by either a fall in serum albumin or increases in

body weight (Fig. 2). As shown in Fig. 4, in patients with

severe VLS, there was a decrease in Fn on day 2 ( 1 day

posttreatment) with little change in albumin levels or in

weight. As shown in Fig. 5, there was also an inverse corre-

lation (r = -0.50, P 0.01) between the maximum concen-

tration of IT achieved and the decrease in Fn bevels observed.

The largest decreases in Fn bevels also correlated with the

presence of the smallest numbers of circulating tumor cells

(r 0.44, P = 0.03). This suggests that a decrease in Fn

bevels may be as predictive a marker for severe VLS as the

absence of circulating tumor cells and serum II concentra-

tions greater than I pg/mb (2, 29, 31). The correlations

between changes in the levels of serum Fn and bevels of II

versus grades of VLS were r -0.38, P < 0.001, and r

0.40, P < 0.001, respectively.

Relationship between Decreases in Serum Fn and

Changes in the Levels of TNF-a. Levels of serum TNF-a

increased in patients with severe VLS (Fig. 6). There was a

trend for correlations between maximal changes in the levels of

Fn in patients with moderate and severe toxicity and increasing

levels of TNF-a (r = -38, P = 0.02).



400

300

200

I 00

0

Fig. 2 Serum Fn levels in patients who

developed severe VLS. The daily levels

of serum Fn were determined in eight

patients who had severe VLS 3-4 daysafter the initiation of IT therapy ($)versus patients with mild VLS (U). The

levels of serum Fn were highest in pa-

tients with severe VLS prior to IT ther-apy. Following treatment with ITs, the

bevels of serum Fn were lower in pa-

tients with severe VLS (mean, 1 15 pg/

ml) than in patients with mild toxicity(195 pg/mb, P < 0.04). Bars, SE.

pretreatment D2 D3 D4

Day


Table 3 Patie nts with inte mipted infusions of ITs due to VLS

Duration of Fninfusions (jig/mi) Fn (pg/mi)(h) or no. prior to IT during IT therapy

Patient Age/sex IT/regimen of doses therapy (daily measurement) Symptoms of VLS

1 60/Fe IgG�RFB4�dgA/CIb 96 241 103, 148, 184, 55 Weight gain 3C, dyspnea, hypotension

2 66/F IgG-RFB4-dgA/CI 72 361 50, 50, 50 Weight gain 3, pulmonary edema, hypotension3 71/F IgG-RFB4-dgA/CI 72 203 52, 53, 52 Weight gain, dyspnea4 58/F IgG-RFB4-dgA/CI 72 139 50, 139, 74 Hypotension, respiratory failure, death

5 6l/M IgG-RFB4-dgAJBI 2 doses 148 50, 50, 55 Pulmonary edema, obiguria, hypotension, death

6 44fF IgG-RFB4-dgAIBI 3 doses 299 94, 94, 241, 208, 94 Weight gain 3, dyspnea, pleural effusion

7 44/M Fab-RFB4-dgAJBI 2 doses 515 500, 94, 139 Pulmonary edema

8 67/F Fab-RFB4-dgA-BI 3 doses 471 139, 241, 188, 188, 500 Dyspnea, oliguria

9 631M IgG-HD37-dgAIBI 2 doses 485 500, 94, 55 Dyspnea, oliguria

10 44/F IgG-HD37-dgAIBI 3 doses 515 130, 241, 241, 299, 188 Weight gain 3, dyspnea

1 1 72/M IgG-HD37-dgA/CI 98 50 50, 50, 50, 50 Weight gain 3, dyspnea, pleural effusion

12 70fF IgG-HD37-dgA/CI 151 188 94, 423, 500, 442, 50, 50 Weight gain 3, dyspnea

a F, female; M, male.b A CI regimen was planned for 192 h. A BI regimen was planned for four doses. The percentage of patients with decreases in the levels of serum

Fn on days 1-3: dose 1, 83%; dose 2, 75%; dose 3, 83%.

C Numbers refer to grade.

E

C0

+1

C0C.)C0C.)

C

C.)C)C

2IL.

DISCUSSION

The major findings to emerge from this study are: (a)

Patients with severe VLS have the highest levels of serum Fn

prior to treatment. (b) Following treatment with ITs, levels of

serum Fn decreased most significantly in patients who devel-

oped severe VLS. (c) The development of severe VLS was

preceded by several days by decreases in the levels of serum Fn,

but not by decreases in the levels of serum albumin or increases

in body weight. (d) The largest decreases in the levels of serum

Fn and the most severe VLS occurred in patients with the

smallest numbers of circulating tumor cells and the highest

levels of serum II. (e) The decreases in the levels of serum Fn

were accompanied by increases in the levels of serum INF-a.

VLS is the major dose-limiting toxicity in patients treated

with RTA-based ITs (1). Manifestations of VLS include fluid

retention, increase in body weight, peripheral edema, pleural

and pericardial effusions, ascites, anasarca, and, in severe form,

renal, pulmonary, and cardiovascular failure. Symptoms are

highly variable among patients for reasons which are not Un-

derstood. During VLS, the levels of serum albumin decrease in

Il-treated patients. In the patients treated with RIA-Ils previ-

ously (2, 29, 31), decreases in albumin ranged from 4 to 40%.

However, the consistency and magnitude of these decreases

were less marked and did not always precede the manifestation

of clinical symptoms. By contrast, the early and rapid decrease

in Fn levels observed in patients with most severe VLS suggest

a causal relationship. Similar early decreases in the levels of

serum Fn have been reported in patients with sepsis, burns,

trauma, and disseminated intravascular coagulation (22, 23, 32).

Persistent decreases in Fn levels in patients with these diseases



-j-�-1--

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A60!

30

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-60

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B60

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Fig. 3 A, changes in the levels of serum Fn in patients with different

grades of VLS. The levels of serum Fn were determined in samplesobtained before therapy and daily during infusion of IT (average of alldays) in 56 patients. The mean Fn levels before therapy ranged from I 75to 300 p.g/ml. The percentage of change versus pretreatment levels ofserum Fn are shown in patients with mild, moderate, and severe VLS.

The decrease in the levels of serum Fn is more significant in patients

with severe VLS than in those with moderate VLS (P < 0.001). The

changes in the levels of Fn in patients with moderate and severe VLS are

significantly different from the changes in patients with mild VLS (P <

0.000001). Means are presented. B, early versus late changes in the

levels of serum Fn. The changes in serum Fn levels after 4 h (s), on thefirst day (0), and on the last day (�) of IT therapy are presented in

patients with mild, moderate, and severe VLS. Significant differences in

the decreases in the levels of serum Fn in severe VLS versus moderateVLS were found on the first day (P = 0.01 ) and the last day (P 0.001)of therapy. Bars, SE.

correlate with organ failure and, in particular, respiratory dis-

tress and cardiac failure. In this regard, the administration of a

Fn cryoprecipitate or purified Fn to patients with decreased

levels of serum Fn frequently reduces the symptoms of VLS

(33-35).

To examine further the relationship between decreased

levels of serum Fn and the development of VLS, we analyzed Fn

levels in patients with different grades of VLS. One problem in

determining this relationship is that there are no “standard”

etiology-based grading criteria for different manifestations of

VLS because of its variable and complex clinical presentation.

Nevertheless, we chose the criteria described by Sausvible et a!.

(2) to define mild, moderate, and severe VLS as discussed in

“Results.” Using these criteria, patients with the most severe

VLS had the highest levels of Fn prior to treatment. Further-

J\J �

I�i I

D2 D3 D4 D5

Day

Fig. 4 Early changes in the levels of serum Fn in severe VLS. Thedaily changes in serum Fn levels (#{149})and albumin levels (A) as well as

weight gain (E) in patients with severe VLS (n = 12) who had

interrupted infusions are shown. Values are expressed as percentage

change versus pretreatment values. The decrease in the levels of serumFn was the earliest event that preceded manifestations of severe VLS.

Means are presented. Bars, SE.

more, the largest decreases in the bevels of serum Fn occurred in

patients whose infusions were interrupted or terminated for any

one of several clinical criteria and who typically had pulmonary

and cardiovascular problems. In these patients, Fn concentra-

tions decreased before clinical manifestations of severe VLS,

thus providing a measurable parameter to predict severe VLS.

Thus, in 83% of the patients with severe VLS, serum Fn levels

decreased during the first day of therapy, and, in 75% of the

patients, the decrease was observed daily during the first 3 days

(Table 3). However, in patients with mild VLS, 35% had de-

creases in the bevels of serum Fn on day 1 and only 14%

maintained this decrease over 3 days. These data suggest that a

persistent decrease in the bevels of serum Fn during the first 3

days of IT therapy might be most predictive for the development

of severe VLS. Furthermore, a concomitant increase in the

levels of serum II might strongly suggest that therapy should be

terminated.

Based on studies of Fn depletion associated with sepsis,

trauma, and other diseases, it has been proposed that as Fn is

consumed by opsonic utilization and rapidly bound to sites of

tissue injury, levels of serum Fn are acutely depleted (17, 22,

23). In our IT-treated patients, the early decreases in the levels

of Fn in the sera of patients who later developed VLS also

suggest a rapid consumption of Fn. This might occur in several

ways: (a) Fn might bind to the RTA portion of the circulating IT

and, as a result, bind less effectively to its receptor on ECs. This

could lead to EC damage by virtue of a disruption of the

extracellular matrix and, subsequently, further consumption of

serum Fn by the injured cells. (b) The II might bind to ECs

nonspecifically, and because these cells are highly sensitive to

RTA, they would be damaged. The damaged cells would con-

sume more Fn and, in addition, be unable to produce and

replenish the Fn. Previous studies have failed to identify a

specific IT-binding site on HUVECs (2 1 ) but such sites could be

present in bow abundance. (c) In either of the above situations,

cytokines released by damaged ECs and/or other tissues could



A

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Fig. 5 Changes in the serum Fn levelsversus levels of IT and circulating tumor

cell levels. A, maximum changes in Se-rum Fn levels correlate inversely withserum IT levels (r = -0.50, P = 0.01).B, changes in the levels of serum Fn onthe first day of therapy correlate with thenumber of circulating tumor cells (r =

0.44, P = 0.03).

1�_0 29.0 39.0 19.0

further increase vascular permeability. Although it is not clear tumor cells further suggest that if circulating tumor cells are

why the higher pretreatment levels of Fn correlate with the most present, they may act as a blood-bone “sink” for the IT and,

severe VLS, it is possible that prior therapies alter the Fn profile hence, decrease its binding to Fn and/or ECs. The damaged ECs

in the sera of some patients, and these patients are in some way in combination with released cytokines and inflammatory me-

predisposed to Il-mediated damage of the vasculature. diators might cause a variety of clinical symptoms. Indeed, it

The correlations between the severity of VLS and both the has been shown that INF-ct and prostaglandins inhibit the

decrease in the levels of serum Fn and the absence of circulating synthesis of Fn (36-39).


Circulating tumor culli (xl 00)



80

mild moderate severe VLS


(I,

a)>a)

ILz

E

a)In

C

a)0�

Ca

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0

Fig. 6 Changes in the levels of serum TNF-a versus severity of VLS.

The percentage of maximum change in the levels of serum TNF-a (E)and Fn (U) in patients with mild, moderate, and severe VLS are shown.

Values are expressed as percentage of change versus pretreatment

values. A significant change in the levels of serum TNF-a (two-tailed,

P = 0.01) was observed in patients with severe VLS as compared tothose with mild VLS. Bars, SE.

With regard to cytokines, we determined whether there

were correlations between changes in the bevels of serum Fn and

TNF-a. TNF-a has been implicated in disrupting the integrity of

vascular endothelium (40-42), and alterations in TNF-a levels

are associated with many other types of tissue damage. Inter-

estingly, it has been reported that Fn has a protective effect on

TNF-a-mediated EC damage (43, 44). TNF-a is considered an

important mediator of fluid extravasation in IL-2 mediated VLS

(45) and it has been shown that administration of pentoxifylline

which is an inhibitor of TNF-a, has a protective effect on VLS

in patients treated with IL-2 (9). Additionally, TNF-a mRNA

expression is up-regulated in the lung, and the administration of

soluble TNF-a receptor diminishes VLS in animals treated with

IL-2 (46). A role for TNF-a in IT-mediated VLS has been

suggested by the demonstration of an increase in TNF-a RNA

levels in mononuclear cells exposed to blocked ricin ITs (47).

The finding that bevels of serum TNF-a increase in the sera of

patients undergoing severe VLS suggests that proinflammatory

cytokines may play a critical role in the pathogenesis of this

condition.

Whatever the sequence of events leading to VLS in IT-

treated patients, our results suggest that early monitoring of

serum Fn levels is helpful in determining which patients are at

risk for severe VLS. Whether or not the administration of Fn to

patients undergoing IT therapy might ameliorate the clinical

manifestations of VLS will depend on knowing why patients

who have the most severe form of VLS also have the highest

levels of serum Fn prior to treatment. Hence, if high levels

predispose patients to VLS, it would not be advantageous to

administer Fn. If, on the other hand, the decreases in Fn levels

predispose to VLS, than Fn therapy should be considered.

ACKNOWLEDGMENTSWe thank C. Patterson, C. Self, P. Lodes, and S. Richardson for

excellent secretarial help.

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2. Sausville, E. A., Headlee, D., Stetler-Stevenson, M., Jaffe, E. S.,

Solomon, D., Figg, W. D., Herdt, J., Kopp, W. C., Rager, H., Steinberg,

S. M., Ghetie, V., Uhr, J., Wifles, R. E., and Vitetta, E. S. Continuousinfusion of the anti-CD22 immunotoxin. lgG-RFB4-SMPT-dgA in pa-

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1996;2:1705-1712. Clin Cancer Res R Baluna, E A Sausville, M J Stone, et al. chain-containing immunotoxins.vascular leak syndrome in patients treated with ricin A Decreases in levels of serum fibronectin predict the severity of

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