Vol. 3, 559-563, April 1997 Clinical Cancer Research 559

Cathepsin D and Dynamic Magnetic Resonance Imaging

Gadolinium Enhancement in Malignant and Benign

Breast Lesions

Paul C. Stomper,1 Janet S. Winston, Steven

Herman, Donald L. Klippenstein, and Leslie E.

Blumenson

Division of Diagnostic Imaging [P. C. S., S. H., D. L. K.] and

Department of Pathology [I. S. W.], Roswell Park Cancer Institute,

School of Medicine and Biomedical Sciences, and Department ofBiomathematics, Roswell Park Cancer Institute and Roswell ParkGraduate Division, Department of Biometry [L. E. B.], StateUniversity of New York at Buffalo, Buffalo, New York 14263

ABSTRACT

Our purpose was to determine whether the expression of

cathepsin D, a proteolytic enzyme implicated in basement

membrane degradation, is associated with dynamic magnetic

resonance imaging (MRI) enhancement of breast lesions. For-ty-five patients with 48 breast lesions underwent gadolinium-

enhanced spoiled gradient recalled echo MIII followed by ex-cisional biopsy and cathepsin D staining and semiquanfitafive

measurement in the lesions. There was no significant difference

in cathepsin D staining of 25 malignant and 23 benign breast

lesions. A significant association was seen between high cathep-

sin D staining and positive axillary lymph nodes in invasive

carcinomas. Nine ofnine (100%) node-positive carcinomas hadhigh cathepsin D, as compared to three of seven (43%) node-

negative carcinomas (P = 0.02). No significant associations

were observed between cathepsin D staining and MRI en-

hancement amplitude, rate, or washout Cathepsin D has noeffect upon MRI gadolinium enhancement of malignant and

benign breast lesions but is associated with positive axillarylymph nodes in invasive carcinomas.

INTRODUCTION

Cathepsin D has been purported to be a prognostic indica-

tor in breast carcinoma (1-12). Cathepsin D is a proteolytic

enzyme that has been implicated in the degradation of basement

membranes. Gadolinium-enhanced breast MR!2 techniques are

being studied to determine whether breast MRI may comple-

Received 8/15/96; revised 12/26/96; accepted 1/3/97.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

indicate this fact.

I To whom requests for reprints should be addressed, at Division ofDiagnostic Imaging, Roswell Park Cancer Institute, Elm and CarltonStreets, Buffalo, NY 14263. Phone: (716) 845-5796; Fax: (716) 845-8759; E-mail: stomper@sc3l02.med.buffalo.edu.

2 The abbreviations used are: MRI, magnetic resonance imaging; MR.magnetic resonance; TR, repetition time; TE, echo time; SPGR, spoiledgradient recalled echo; DCIS, ductal carcinoma in situ.

rnent mammography in the detection of carcinoma or aid in the

differentiation of benign from malignant lesions (13-25). Because

delivery of intravascular agents to tumors requires transport of the

agent across the vascular endotheiurn and basement membrane to

the interstitiurn and cells of tumors, it is a reasonable hypothesis

that cathepsin D levels in breast lesions may be associated with

MRJ contrast enhancement. We know of no studies in the literature

that assess possible association between cathepsin D immunohis-

tochemical staining and MRI enhancement of breast lesions. This

study was undertaken to assess associations between cathepsin D

staining of malignant and benign breast lesions and dynamic M�

parameters of amplitude, rate, and washout.

MATERIALS AND METHODS

This study population consists of a subgroup of 45 patients

from a previously reported single-institution dynamic contrast-

enhanced breast MRI trial (22), who underwent excisional bi-

opsy and who also had cathepsin D staining of histological

sections of each lesion. Each of the patients in this study (a) had

a suspicious lesion detected by mammography and/or clinical

examination, (b) volunteered and gave written informed consent

for the Institute’s research review board-approved trial, (c)

underwent dynamic contrast-enhanced MRI within 2 weeks of

the diagnostic mammogram and clinical examination, and (d)

underwent excisional biopsy of the lesion within 2 weeks of the

breast MRI examination. The median age of the patients was 54

years (range, 26-82). Eighteen (40%) patients were 49 years or

younger; 27 (60%) patients were 50 years or older.

The study population consisted of 26 lesions detected by

mammography only, 13 lesions detected by mammography and

clinical examination, and 9 lesions detected by clinical exami-

nation only in patients with normal mammograms. Twenty-six

of the mammographic lesions were soft-tissue abnormalities; I 3

were calcifications. The median size of the mammographic

lesions was 13 mm (range, 5-60 mm). The median size of the

clinically palpable lesions was 20 mm (range, 10-70 mm).

Breast MRI was obtained using a 1.5-I MRI clinical scan-

ner (Signa; GE Medical Systems, Milwaukee, WI) and a corn-

rnercially available GE breast coil (model no. M1O8SBR). This

radio-frequency coil is a receive-only set of coils that permits

the operator to select image acquisition from one or both breasts

per data set. After axial localizer images were obtained, the

involved breast was scanned in the sagittal plane using an 1 8-cm

field of view, 3-mm section thickness, and 0-mm intersection

gap. The T1-weighted images were obtained with a IR of 651

ms, TE of 12 rns, 256 X 192 matrix, and one acquisition. Fast

spin-echo T2-weighted images were obtained with a IR of 6000

ms, TE of 120 ms, 256 X 128 matrix and two acquisitions. On

the basis of the T1-weighted and 1,-weighted images, five

contiguous 3-mm sections were selected in the center of the

lesion for SPGR pre- and postcontrast scanning using a IR

Research. on June 27, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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� ,v�Fig. I A. precontrast SPGR MR image. B, postcontrast SPGR MR image obtained I mm after contrast injection shows enhancement of 2.5-

cm-diameter mass (arrow). The amplitude of enhancement was 4.7 post-/precontrast. Histological examination showed invasive ductal carcinoma. C,high-power photomicrograph ( X400) shows dark intracytoplasmic staining of lysosomes for cathepsin D in both invasive carcinoma and DCIS.

Cathepsin D staining was present in 20-50% of cells (2+). Axillary lymph node dissection was negative (0 of 10 nodes).

560 Cathepsin D and Breast MRI Enhancement

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Clinical Cancer Research 561

Table I Cathepsin D staining of malignant and benign breastlesions”

Subgroup

(No. of lesions)

cathepsin D staining

2 + or less 3 +

All lesions 14 34All malignant lesions 8 17

Invasive carcinomab 7 15DCISC 1 2

All benign lesions 6 17Fibroadenomas 0 6

Other benign lesions 6 1 1

U There were no significant associations between cathepsin D stain-

ing and histological diagnosis.

b Two of three lobular carcinomas stained 3+; all others wereductal.

C Two cases had microscopic (less than 1 mm) foci of invasion.

of 12.6 ms, TE of 3.6 ms, 60#{176}flip angle, 256 X 192 matrix,

and two acquisitions. Gadopentetate dimeglurnine (Magnevist;

Berlex Laboratories, Wayne, NJ) was administered by hand as a

bolus injection at a dose of 0. 1 rnmol/kg over 10 s followed by

a 20-rnl saline flush via extension tubing and a 20-gauge cath-

eter in an antecubital vein while the patient was in the magnet,

obviating the need for any patient movement between scans.

Scanning began immediately following the injection, and serial

dynamic images were obtained every 30 s for each of the five

contiguous image sections over S mm.

All breast MR images were interpreted jointly by three radi-

obogists with knowledge of the mammographic and clinical find-

ings but without knowledge of the histological findings. Matching

film sets of pre- and postcontrast images were displayed and

interpreted side by side. Two-mm2 region of interest signal inten-

sity measurements were made in two areas of maximal enhance-

ment in the lesion as determined by visual inspection of the dy-

namic MRI scans. The region of interest showing the highest

amplitude of enhancement in the lesion was used for the MR

enhancement time-intensity curves. When tumor enhancement was

not apparent by visual inspection of the MRI scans, lack of tumor

enhancement in each case was confirmed by construction of com-

puter-assisted pixel-by-pixel subtraction images. The three-param-

eter mathematical model used in earlier studies (20, 22) was fitted

to the MR enhancement time-intensity curves using the equation:

SI(t) = I + A[1 - exp(-t/Tc)] - Ct

where SI(t) is the normalized signal intensity (signal intensity

observed at time t divided by the precontrast signal intensity) at

postcontrast time 1, A is the enhancement amplitude, Tc is the

time constant for arrival of contrast material, and C is the

first-order washout rate. Postcontrastlprecontrast signal inten-

sity ratios for each time interval during dynamic scanning were

determined using the predicted curve.

Histological Evaluation. Histological material derived

from excisional biopsy was stained with H&E. The histological

interpretations were performed by one pathologist (J. S. W.)

without knowledge of the MRI findings. For the purposes of this

study, histological diagnoses were categorized as (a) invasive

carcinoma including invasive ductal or invasive lobular carci-

Table 2 Association between cathepsin D staining and size andnodal status of invasive carcinomas

Ca thepsin D staining

P value2+ or less

Pathological size

1-10mm 3 5

11-20mm 4 7

Greater than 20 mm I 5 NS”Nodal status”

Negative nodes 4 3

Positive nodes 0 9 0.02

“ NS. not significant.

I, Nodal status of 16 patients who underwent axillary lymph node

dissections.

noma; (b) DCIS, including pure DCIS and DCIS with micro-

scopic (< I mm in diameter) foci of invasion, because it was

uncertain whether the microscopic foci of invasion were in-

cluded in the limited region of dynamic scanning; (c) fibroad-

enomas; or (d) other benign lesions.

Cathepsin D Immunohistochemical Staining. After re-

view of each case, the most representative block of the suspi-

cious lesion was selected. From each of the selected blocks,

several consecutive 4-p.m sections were obtained. One section

was stained by immunohistochemical technique for cathepsin D

antigen. Staining was performed on the Ventana 320 Automated

Slide Staining System (Ventana Medical Systems, Tucson, AZ).

The detection of cathepsin D utilized a mouse monoclonal

antibody (Oncogene Science) at a dilution of I :200 and the

avidin-biotin complex technique following predigestion with

protease. Diaminobenzidine was used as a chromogen, and the

sections were then counterstained lightly with hematoxylin.

Cathepsin D staining was assessed by estimating the percent-

age of positively staining cells within each lesion examined. The

amount of staining is indicated as 1 +, 2+, or 3+, where I +

represents fewer than 20% positive cells, 2+ represents 20-50%

positive cells, and 3 + represents greater than 50% positive cells.

Analysis. To identify any association between the ca-

thepsin D staining and MRI enhancement features, the following

features were compared. For dynamic contrast-enhanced MRI,

the following categorization of lesions with greater enhance-

ment parameters was used: (a) greater amplitude: lesions that

reached postcontrast/precontrast enhancement ratios equal to or

greater than the median value of 3.0 within 2 mm; (b) greater

rate: lesions that had a time constant for arrival of contrast

(parameter Tc) less than or equal to the median of 20; and (c)

greater washout: lesions that during the 5-mm dynamic scanning

had a washout rate (parameter C) equal to or greater than the

median of I .0 X l0�. Cathepsin D immunohistochemical

staining of lesions was categorized as 3+ or 2+ or less.

The Fisher exact text was used to test the association in the

2 X 2 tables constructed using MRI enhancement amplitude (A)

less than 3 versus amplitude greater than or equal to 3, ampli-

tude less than 2 versus amplitude greater than or equal to 2, rate

(Tc) greater than 20 versus rate (Tc) less than or equal to 20, or

washout (C) less than or equal to 0.001 versus washout (C)

greater than 0.001 (26). P values less than 0.05 were considered

significant.

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562 Cathepsin D and Breast MRI Enhancement

Table 3 Associations between cathepsin D staining an d MRJ enhancement parameters

Cathepsin D staining (No. of lesions)Malignant lesions with

positive nodesAll lesions#{176} All malignant lesions”

MRI enhancement 2+ or less 3 + 2+ or less 3 +

All benign lesions”

2+ or less 3 + 2+ or less 3 +

Amplitude (A)Less than 3 9 20 3 10 6 10 0 4Greaterthanorequalto3 5 14 5 7 0 7 0 5

Lessthan2 4 1 1 0 2 4 9 0 0

Greater than or equal to 2 10 23 8 15 2 8 0 9Rate (Tc):

Greater than 20 8 22 3 10 5 12 0 3Less than or equal to 20 6 12 5 7 1 5 0 6

Washout (C)

Less than or equal to 0.001 5 17 3 9 2 8 0 4Greater than 0.001 9 17 5 8 4 9 0 5a No significant associations were found.

RESULTS

Positive cathepsin D staining was noted in both benign and

malignant epithelial cells and macrophages and was located in

intracytoplasmic granules, most likely corresponding to lyso-

somes and endosomes (Fig. 1). In general, benign epithelium

showed a low-intensity small granular staining, whereas in situ

and invasive carcinomas and macrophages displayed a more

consistent, diffuse, and higher-intensity staining of larger gran-

ules. Seventeen of 25 (68%) malignant and 17 of 23 (74%)

benign lesions showed 3+ (greater than 50%) positivity staining

cells for cathepsin D. These results are summarized in Table 1.

The associations between cathepsin D staining and size and

nodal status of the invasive carcinomas included in the study is

shown in Table 2. There was no significant association between

cathepsin D staining and pathological size. There was a significant

association between high cathepsm D staining and positive axillaty

lymph nodes. Of 16 patients who underwent lymph node dissec-

tion, 9 of 9 (100%) patients with positive lymph nodes had high

(3+) cathepsin D staining of their primary tumors as compared to

3 of 7 (43%) patients with negative lymph nodes (P = 0.02).

The associations between MRI enhancement parameters

and cathepsin D staining are shown in Table 3. No significant

associations were observed between cathepsin D staining and

the MRI enhancement parameters of amplitude greater or equal

to three, amplitude greater or equal to two, rate, or washout.

DISCUSSION

Our study shows no significant association between Ca-

thepsin D staining and MR gadolinium enhancement amplitude,

rate, or washout of malignant and benign breast lesions. How-

ever, a significant association was seen between cathepsin D

staining and positive axillary lymph nodes in invasive carcino-

mas. Nine of nine (100%) node-positive carcinomas had high

cathepsin D as compared to three of seven (43%) node-negative

carcinomas (P = 0.02).Cathepsins are ubiquitous lysosomal enzymes that are clas-

sified both functionally and according to their active site. In the

metastatic process, these proteolytic enzymes play a role in

mediating passage of malignant cells between tissue and vessels

via degradation of the basement membrane. As reviewed by

Schwartz (2), measurements of cathepsin D in breast cancer

have been shown in a majority of studies to be significant in

predicting recurrence and may also predict disease-free and

overall survival. A potential role of cathepsin D in models

utilizing histological and molecular features of tumors in the

hope of obtaining similar prognostic information as axillary

lymph node dissection warrants continued investigation (27-

29). Reported differences concerning the role of cathepsin D as

a prognostic marker in breast cancer may be related in part to the

methodology used and the employment of assays of antibodies

prepared to different portions of the molecule (2, 4).

The use of MR enhancement parameters as an in vivo func-

tional assay and predictor of biological and prognostic features of

malignant and benign breast lesions has been a topic of several

recent reports. In a prior report including 22 invasive carcinomas,

we have shown no significant association between MR gadolinium

enhancement parameters and tumor size, nodal status, or grade

(22). An association between a peripheral pattern of enhancement

in invasive carcinomas and high DNA S phase has been shown

(23). Several studies of vessel density determined by factor Vifi

staining have shown that MRI gadolinium enhancement is not

highly predictive of vessel density (24, 25).

Although basement membrane integrity would appear to

play a role in any transport mechanism between vessels and

surrounding tissue, its relative importance may depend on the

size and transport mechanism for a given molecule or cell. Jan,describing barriers to drug delivery in solid tumors, has de-

scribed multiple factors involving fluid and molecular transport

in the delivery of intravascular agents through the endothelium

and basement membrane into the interstitium and cells of le-

sions (30, 31). Macromolecular MRI contrast media currently

being studied in animal models may be more suited than the low

molecular weight gadolinium to define hyperpermeability asso-

ciated with high cathepsin D levels (32-34). Further develop-

ment of contrast-enhanced breast MRI and analysis techniques

may also improve our ability to perform in vivo assays of vessel

hyperpermeability in lesions.

We conclude that, although there is an association between

high cathepsin D staining and positive axillary lymph nodes in

invasive carcinomas, there is no significant association between

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Clinical Cancer Research 563

cathepsin D staining and MR low molecular weight gadolinium

enhancement amplitude, rate, or washout in malignant and be-

nign breast lesions. Factors other than basement membrane

integrity, including hemodynamics and capillary permeability

and transport factors, are probably more responsible for the

range of gadolinium enhancement seen in breast lesions.

ACKNOWLEDGMENTS

We gratefully acknowledge Carol Kaminski, R.T., and Jeanne Cervi,

R.T.; Joyce Bailey for performing immunohistochemical staining; and

Christine Sheehan for data management and manuscript preparation.

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