the small heat shock protein hsp27 is not an independent … · vol. 2, 1199-1206, july 1996...

Vol. 2, 1199-1206, July 1996 Clinical Cancer Research 1199

The Small Heat Shock Protein HSP27 Is Not an Independent

Prognostic Marker in Axillary Lymph Node-negative Breast

Cancer Patients’

Steffi Oesterreich, Susan G. Hilsenbeck,

Daniel R. Ciocca, D Craig Allred,

Gary M. Clark, Gary C. Chamness,

C. Kent Osborne, and Suzanne A. W. Fuqua2

Department of Medicine, Division of Medical Oncology [S. 0..

S. G. H.. G. M. C., G. C. C., C. K. 0., 5. A. W. F.], and Department

of Pathology ID. C. A.]. University of Texas Health Science Center at

San Antonio, San Antonio, Texas 78284-7884. and Laboratonio de

Reproduccion y Lactancia, LARLAC. CC. 855. 5500 Mendoza,

Argentina [D. R. C.]

ABSTRACT

Heat shock protein 27 (hsp27) belongs to the family of

heat shock proteins and is thought to be involved in then-

motolenance, cell proliferation, drug resistance, and chaper-

one processes. The aim of this study was to investigate

whether hsp27 levels are correlated with clinical outcome in

axillary lymph node-negative breast cancer patients. We

describe a Western blot study measuring hsp27 levels in 425

patients and an immunohistochemistry (IHC) study analyz-

ing 788 patients. Results obtained by both methods were

concordant. Univaniate survival analysis was performed

considering hsp27 either as an optimally dichotomized van-

able or as a continuous variable. Additional data include age

at biopsy, tumor size, estrogen receptor (ER) and progester-

one receptor status, tumor ploidy and percentage of cells in

S phase, and adjuvant therapy. hsp27 levels correlated pos-

itively with ER status (P = 0.0001 in Western blot and IHC

study), progesterone receptor status (P = 0.0001 in Western

blot and IHC study), and aneuploidy (Western blot study, P

= 0.0012; IHC study, P 0.0004) but not with tumor size

(Western blot study, P 0.69; IHC, P 0.53) or S phase

(Western blot study, P = 0.19; IHC study, P 0.38). Over-

all, there was no relationship between hsp27 expression and

disease-free survival (Western blot study, P 0.70/0.54;

IHC, P = 0.47/0.30) or overall survival (Western blot study,

P = 0.16/0.15; IHC, P 0.46/0.78). Exploratory subset

analyses defined by ER status and use of adjuvant treatment

indicated that in ER+/untreated patients, high hsp27 levels

correlated modestly with shorter disease-free survival

Received I 2/2 1/95; revised 4/5/96: accepted 4/1 1/96.

� Supported by Specialized Programs on Research Excellence Grants

P50 CA58183. P30 CA54174. and P01 CA30195 and in part by

CONICET/Governrnent of Mendoza (Argentina).2 To whom requests for reprints should be addressed. at Department of

Medicine. Division of Medical Oncology, University of Texas Health

Science Center at San Antonio. 7703 Floyd Curl Drive, San Antonio,

TX 78284-7884. Phone: (210) 567-4787: Fax: (210) 567-6687.

(Western blot, P 0.04/0.04; IHC, P 0.1 1/0.03). hsp27 is

not a useful prognostic marker for the clinic in axillary

lymph node-negative patients. However, the finding of mod-

est prognostic value of hsp27 in the subgroup of ER+/

untreated patients raises new questions about the biological

function of hsp27 in breast cancer.

INTRODUCTION

hsp273 belongs to the family of heat shock proteins (re-

viewed in Ref. I). A variety of different functions have been

described for hsp27, including a role in thermotolerance (2), cell

proliferation, drug resistance (3). adtin polymerization (4, 5),

and chaperone processes (6, 7). In breast cancer cells, hsp27 was

originally identified as an estrogen-responsive gene (8. 9). The

expression of hsp27 is very high in normal estrogen-sensitive

target organs, such as breast, uterus, placenta. fallopian tubes,

vagina, and skin ( 10), but varies widely in breast cancer ( 1 1).

hsp27 has been studied as a prognostic marker in several

tumor types including prostate and bladder cancer, malignant

fibrous histiocytoma, and neuroblastoma (12-14). Of seven

reported studies in breast cancer using different assay methods

and patient populations (Table 1 ), three reported that high ex-

pression of hsp27 is a marker for worse outcome (15-17), two

showed no correlation with clinical outcome (18, 19), and two

studies indicated an association with improved outcome (20,

21 ). In view of these partially contradictory results, we decided

to repeal our original pilot study (15) using two different nieth-

ods in a larger, independent group of patients. We focused on a

node-negative population, most of whom did not resume adju-

vant therapy. Since most axillary lymph node-positive patients

will receive adjuvant therapy independent of the status of other

tumor markers, the challenge is to identify a subset of axillary

lymph node-negative patients who can forego adjuvant therapy.

In this study, we describe a Western blot analysis meas-

uning hsp27 levels in 425 axillary lymph node-negative patients

and an IHC study analyzing 788 node-negative patients. Ele-

vated hsp27 levels correlate with a shorter DFS in ER+/un-

treated (surgical intervention, but no adjuvant treatment) pa-

tients but are not associated with DFS survival or overall

survival in the whole patient population. Therefore, hsp27 is not

a useful, independent prognostic marker in axillary lymph node-

negative patients.

3 The abbreviations used are: hsp27. heat shock protein 27: ER. estrogen

receptor: PgR. progesterone receptor: IHC. irnrnunohistochernistry:

DFS. disease-free survival; OS. overall survival.

Research. on October 29, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

1200 hsp27 Is Not an Independent Marker in Node-negative Breast Cancer

Table 1 Prognostic significance of hsp27 in breast cancer patients

Association with

Investigators (Ref.) ii Follow up (rno) Nodal status Assay DFS OS

Tandon et al. (15) 198

161

81

68

Neg.”

Pos.

Western blot

Western blot NS

NS

NSThor ci’ al. (16) 291

(total)

92 Neg. IHC NS NS

1-3 Pos. nodes�4 Pos. nodes

IHC

IHC

(a = 66)

NS

NS

NS

Love and King (17) 361 74 Pos. vs. neg. IHC tin node neg.

patients: ii =

NS

Darrnstrup et al.(18) 103 ? Mixed IHC NS NS

Tetu ci’ al. (19) 890 78 Pos. IHC NS NSHurlirnan et al. (20) 196 81 Mixed IHC I “

Seymour et al. (21) 51 ? Pos. IHC ? I

(1 Neg.. negative: Pos., positive: NS, nonsignificant; � , decreased: I � increased: ?. unknown.

I, The survival was measured at 5 years after diagnosis: borderline significance was reached in ER-negative tumors.

PATIENTS AND METHODS

Patient Population

The patients in this study were identified from a large

database of patients who had hormone receptor assays per-

formed in our laboratory and comprise a total of 903 axillary

node-negative breast cancer patients diagnosed and resected

between 1974 and 1988. Selection criteria included presentation

with primary breast cancer treated with mastectomy or lumpec-

tomy, with or without postoperative radiation therapy, and ab-

sence of involvement of axillary lymph nodes or any distant

metastasis. Available clinical data included age at biopsy (diag-

nosis), tumor size, ER and PgR status, and tumor ploidy and

percentage of cells in S phase, as determined by flow cytometry

(22, 23), and adjuvant therapy. Patients were followed for dis-

ease recurrence and survival as described previously (24). The

Western blot study was done first and includes 425 cases. Three

hundred ten of these samples were later analyzed by IHC;

however, for some cases, no paraffin sections were available.

The IHC study was extended by using an additional 478 cases,

analyzing a total of 788 cases by IHC. The median follow-up

from diagnosis for unrelapsed cases was 7 1 and 53 months for

the Western blot and IHC studies, respectively.

hsp27 Analyses

Western Blot. Breast tumor specimens were frozen in

liquid nitrogen immediately after excision and stored at -70#{176}C.

Breast tumor tissues were fractured in the frozen stale, and an

average of 50 rng of pulverized tissue was used from each case.

Extracts from pulverized frozen tumors were prepared, and 100

�ig of protein were subjected to Western blot analysis as de-

scnibed previously (25, 26). The primary antibody was a mono-

clonal antibody directed against human hsp27, which was de-

veloped in our laboratory (27). It was used at a dilution of

1 : 1000 in blocking buffer. After washing, the blots were incu-

baled for 2 h with ‘251-labeled anti-mouse antibody (100,000

cpm/ml; Amersham Corp. Arlington Heights, IL). Each poly-

acrylamide gel contained an internal protein standard from

human MCF-7 breast tumor cells. Evidence of hsp27 protein

degradation, such as multiple faster migrating bands, could not

be detected (data not shown). The hsp27 bands were quantitated

by densilomelnic scanning, as described elsewhere (15). The

levels of hsp27 were calculated in arbitrary units per 100 �ig of

protein by the ratio of the integrated densitometry signal in the

tumor samples relative to the MCF-7 standard on each gel.

IHC. IHC was performed on frozen, partially pulverized,

small breast carcinoma samples prepared for paraffin sections as

reported elsewhere (28). Briefly, frozen pulverized tissue was

rehydraled at room temperature in phosphate buffer and fixed

for 4 h in 10% formalin; then the particles were concentrated in

warm agar. After embedding in paraffin wax, tissue sections (5

p.m thick) were used to evaluate hsp27 expression by IHC (29),

using a commercial avidin-biotin-peroxidase method (Vector

Laboratories, Burlingame, CA). A quality control examination

was performed on each sample (stained with H&E) to exclude

those samples with an inadequate number of malignant cells,

unusual cellular damage, or necrosis. Diaminobenzidine was

used for developing color; methyl green was used for counter-

staining. Each run included positive control sections from

MCF-7 cells, and negative controls were run by omitting the

primary antibody. Immunostained slides were examined by light

microscopy, and a positive signal was defined as the presence of

distinct staining in the tumor cell. The signal was semiquanti-

latively scored estimating the proportion of positively staining

tumor cells on the entire slide (0, none; 1 , less than one-

hundredth; 2, one-hundredth to one-tenth; 3, one-tenth to one-

third; 4, one-third to two-thirds; 5, greater than Iwo-thirds) and

the average intensity of staining (0, no staining; I , weak slain-

ing; 2, moderate staining; 3, strong staining). Intensity and

proportion scores were added to obtain the final total score. This

scoring system has been successfully applied to evaluate steroid

receptors and other prognostic factors in previous studies (30,

31).

Statistical Analyses

Statistical analyses focused on Iwo issues: (a) the associ-

ation between hsp27 measurements and other clinical variables;

and (b) the prognostic value of hsp27. Associations between

hsp27 values and other clinical variables were first examined



Clinical Cancer Research 1201

Table 2 Summary of patie nt characte nistics and associations with hsp27 level s measured by West em blot or IHC

Western blot study IHC study

hsp27 association hsp27 association

Groups F,” % -Ysp/, P ii” % -y,� P

All Cases 424 100 788 100

Age �50 yrs

>50 yrs

1 19

305

28.1 0.08 0.09

71.9

191

597

24.2

75.2

0.09 0.60

ER Negative

Positive

99

325

23.3 0.34 0.0001

76.7

159

627

20.2

79.8

0.18 0.0001

PgR NegativePositive

186219

45.9 0.19 0.000154.1

310

457

40.4

59.6

0.15 0.0001

Tumor size �2 cm

>2 cm

156

268

36.8 0.02 0.69

63.2

410

355

53.6

46.6

0.02 0.53

Ploidy Diploid

Aneuploid

141

268

34.5 0.0012’

65.5

307

474

39.3

60.7

0.0004’

% S Phase <6.7%�6.7%

201

170

54.2 0.07 0.19

45.8

404

282

58.9

41.1

0.03 0.38

Endocrine therapy No

Yes

317

92

77.5 0.94’

22.5

653

89

88

12

0.63’

Chemotherapy No

Yes

357

52

87.3 0.38’

12.7

677

68

90.9

9.1

0.02’

“ Not all variables were measurable in all cases.

I’ � Spearman’s rank correlation, using actual values.C Groups compared by two-sample Wilcoxon rank sum test.

using Spearman rank correlations for continuous clinical van-

ables (tumor size, ER, PgR, and percentage in S phase) and then

using Wilcoxon rank sum statistics to compare distributions of

hsp27 for categorical variables (ploidy, endocrine therapy, and

chemotherapy). Two prognostic outcomes were examined: DFS,

defined as the time from diagnosis to first recurrence, or last

contact; and OS, defined as the time from diagnosis to death

(from any cause), or last contact. Survival times (DFS or OS)

longer than 10 years were truncated at 1 0 years and censored to

reduce the influence of a small number of cases with very long

follow-up. In prognostic analyses, clinical variables were di-

chotomized as follows: age at diagnosis, �50 versus age >50

years; tumor size, �2 cm versus >2 cm; ER, <3 versus

fmol/rng protein; PgR, <5 versus �5 frnollmg protein; diploidy

(DNA index, 1 .0) versus aneuploidy; percentage of S-phase

fraction, s6.7 versus >6.7% (24); no adjuvant endocrine ther-

apy versus endocrine therapy; and no adjuvant chemotherapy

versus chemotherapy. Univaniale survival curves were estimated

using the method of Kaplan and Meier and compared using the

log-rank test (32). Culpoinl analysis with P adjustment (33) was

used to dichotomize hsp27. In cutpoint analysis and related

methods that use repealed testing of the data set to select an

optimal cutoff value, type I errors (false positives) increase

with the number of culpoints examined (34), and P adjustment

(Padjusted) is necessary to correct for this. Cox proportional

hazards regression (32) was used to examine the prognostic

value of hsp27 as either a dichotomized or continuous

log-transformed variable.

RESULTS

Association of hsp27 Expression with Other Prognostic

Factors. General patient and tumor characteristics are shown

in Table 2. Four hundred twenty-five samples were examined by

Western blot analysis, and 788 samples were analyzed by IHC

for hsp27 expression; representative experiments of the Western

Blot analysis, as well as the IHC assay, are shown in Fig. 1 . The

Western Blot shows different hsp27 levels in 10 breast tumors

and the internal protein standard from MCF-7 cells. The IHC

reveals a strong cytoplasmic signal in breast cancer cells.

Complete data on all the measured prognostic variables

were available on 346 and 613 cases, respectively. The majority

of these node-negative patients did not receive endocrine ther-

apy (77.5 and 88% in the Western blot and IHC studies, respec-

tively) or chemotherapy (87.3 and 90.9%, respectively). In the

Western blot study, hsp27 expression correlated with the ex-

pression of the hormone receptors ER (P = 0.0001) and PgR

(P = 0.0001) but not with tumor size (P 0.69) or percentage

in S phase (P = 0. 19). Aneuploid tumors tended to have higher

hsp27 values (P = 0.012). Similar results were obtained for the

tumor specimens analyzed by IHC. In general, results obtained

by both methods, Western blot and IHC, were concordant (�y�

= 0.505; P = 0.0001). Again, hsp27 expression as obtained

with IHC correlated with ER (P = 0.0001), PgR (P = 0.0001),

and aneuploidy (P = 0.0004) but not with tumor size (P = 0.53)

or S phase (0.38).

Clinical Outcome and Expression of hsp27. Univariate

survival analyses are summarized in Table 3. In the Western blot

datasel, small tumor size and a low percentage in S phase were

significantly related to longer DFS and OS, whereas age over 50

was related to longer OS. In the larger IHC dataset, which had

shorter follow up (53 months versus 71 months) and smaller

tumors, we found that age over 50 years, small tumor size, low

percentage in S phase, and diploidy were all associated with a

longer DFS. Only tumor size and S phase were related to OS.

In the Western blot dataset, there was no detectable rela-

tionship between hsp27 expression and either DFS or OS (Table

3), regardless of whether hsp27 was considered as an optimally

dichotomized variable (P�,djusted 0.70 or 0. 16 for DFS and OS,




Tumors MCF-7

1 2 3 4 5 6 7 8 910 �

� :lj�� ,�* ;�;:�#{248}�

Fig. 1 Western Blot (left) and IHC (right) using hsp27-specific antibodies. The Western Blot shows the hsp27 level in 10 breast tumors and the

internal protein standard (100, 50, and 25 �g) from MCF-7 cells.

Table 3 Univari ale su rvival analysis of all p atients an d all variables

DFS#{176} 0 5�

Western Western

blot study IHC study blot study IHC study

HRb p HRb p HRb p HR” p

Age 1.12 0.6 1.33 0.12 0.61 0.04 0.83 0.29ER 0.71 0.11 0.82 0.27 0.89 0.61 0.85 0.37PgR 0.92 0.67 0.75 0.06 0.79 0.26 0.79 0.14

Tumor size 1.95 0.003 2.56 0.0001 1.53 0.05 1.83 0.0004% S Phase 1 .55 0.03 1 .55 0.007 1 .56 0.04 1 .46 0.03Ploidy 1.24 0.31 1.78 0.0006 1.31 0.23 1.27 0.17hsp27c 0.70 0.47 0.16 0.46hsp27” 1.07 0.56 1.25 0.29 1.18 0.16 1.07 0.77

a Log-rank results.

b HR. hazard ratios from Cox regression with a single covariate. For each variable, the reference group is the first group listed for that variable

in Table 2.C p� for hsp27 are from cutpoint analyses and are corrected for multiple comparisons. Hazard ratios could not be determined.

d Ps for hsp27 are from Cox regression where log(hsp27 + 1) was the only explanatory variable. Hazard ratios represent the effect of a 10-fold

increase in hsp27 score.

respectively) or as a log-transformed continuous variable (P =

0.54 and 0. 15, respectively). Given the sample size of this

dataset, the rate of censoring, and the modest correlation of

hsp27 with other variables of known prognostic value, we

estimate that a moderate independent prognostic value (a hazard

ratio of 2.0 or more) for hsp27 would have been detectable with

more than 85% power at the 5% level of significance. Likewise,

in the IHC dataset, there was no detectable relationship between

hsp27 and either DFS or OS, regardless of whether hsp27 was

considered as an optimally dichotomized variable (�adjusted

0.47 and 0.46, respectively) or as a continuous log-transformed

variable (P = 0.30 and 0.78, respectively). Again, given the

sample size, rate of censoring, and the correlation of hsp27 with

other variables, a moderate independent prognostic value for

hsp27 would have been detectable with more than 95% power at

the 5% level of significance. Survival curves (shown in Fig. 2)

clearly confirm these findings. The curves for low versus high

hsp27 expression are nearly overlapping for DFS as well as OS.

Umvariate Survival Analysis in ER+IUntreated Pa-tients. Since our datasets are fairly large, we decided to per-

form subgroup analysis of the tumors. Subgroups were defined

by ER status and use of adjuvant treatment (ER+/treated,

ER+/untreated, ER-/treated, and ER-/untreated). These

groups are of interest for several reasons: (a) it was recently

shown that hsp27 can be induced by different cylotoxic drugs

commonly used in the treatment of cancer patients, including

daunomycin, doxorubicin, 3 ‘ -fluorodeoxythymidine, and

vincnisline (35). Furthermore, hsp27 itself may be involved in

drug resistance, e.g. , overexpression of hsp27 results in de-

creased sensitivity to doxorubicin in breast cancer cells (3).

(Unfortunately, direct influence of hsp27 expression on the

effect of doxorubicin treatment in patients could not be

evaluated in this study, since the few treated patients received

various nonstandardized treatments); and (b) hsp27 is an

estrogen-regulated gene, and its expression correlates with

ER expression in a range of estrogen-sensitive cells, includ-

ing primary breast cancer (36). We repeated the univaniale

survival analyses in each of the four subgroups (Table 4). In

ER+/untreated patients, but not in the other subsets, a weak

correlation between hsp27 clinical outcome was found (Table

4). In the Western blot datasel, hsp27 expressed levels once

considered either as an optimally dichotomized variable



I� hsp27 (n=�3)

p=O.58 High h8p27 (n=211)

A0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

B0.9

0.8

0.7

0.6

0.5

High hsp27 (n’=310)

Low hsp27 (n=478)

C1.o0.9

0.8

0.7

0.6r)� 0.5

0.4

0.3

0.2

0.1

0.0

D1.o0.9

0.8

0.7

0.6

C,,0 0.5

0.4

0.3

02

0.1

0 24 48 72 96 120

Months

� � hsp27 (n=�3)

p=O.22 High hsp27 (n=211)

0 24 48 72 96 120

Months

� L� hsp27 (n=478)

High hsp27 (n=310)

p=0.13U)0

_________________________________ 0.0

0 24 48 72 96 120

Months

Fig. 2 Survival curves (OS and DFS) of all patients as a function of hsp27 expression measured by Western blot (A and B) or IHC (C and D). Thegraphs are based on dichotomization of hsp27 at median values.


patients are shown in Fig. 3.

0.4

0.3

0.2

0.1

0.0

0 24 48 72 96 120

Months

p=O.44

Table 4 Univaniate survival analysis in ER+/untreated patients considering hsp27 either as an optimally dichotomized or as a

continuous variable

DFS” OS”

Western

blot study IHC study

Western

blot study IHC study

HR” P HR’ P HR” P HR” p

hsp27’

hsp27” 1.37

0.04

0.04 1.32

0.1 1

0.03 1.17

0.52

0.30 1.17

0.15

0.22

“ Log-rank results.

I’ HR. hazard ratios.‘� PS for hsp27 are from cutpoinl analyses and are corrected for multiple comparisons. Hazard ratios could not be determined.‘I p� for hsp27 are from Cox regression where log(hsp27 + 1 ) was the only explanatory variable. Hazard ratios represent the effect of a 2.7-fold

increase in hsp27 score.

(13�tdjusted = 0.04) or as a continuous variable (using log-transform

P = 0.04) was modestly associated with DFS but was not associ-

ated with OS. Analogous results were obtained with the IHC

dataset; hsp27 expression considered as a continuous variable (us-

ing a log-transform) was associated with DFS (P = 0.03) but not

with OS (P = 0.22). The curves for DFS and OS of ER+/untreated

DISCUSSION

The heat shock or stress-response proteins are a family of

intracellular proteins with many functions, one of which is to

protect cells from a variety of external toxic stimuli (reviewed in

Ref. 1). We first identified hsp27 in breast cancer cells as an

estrogen-regulated protein that is overexpressed in some human

breast cancer specimens (8, 9). We hypothesized that hsp27,



p=0.03

I� hsp27

High hsp27 (n=105)

p-0.03

Low hsp27 (n=106)

High hsp27 (n=105)

A i.o

0.9

0.8

0.7

0.6

� 0.5

0.4

0.3

0.2

0.1

0.0

B

0.7

0.6

� 0.5

0.4

0.3

0.2

0.1

0.0

0 24 48 72 96 120

Months

C1.o0.9

0.8

0.7

0.6U)� 0.5

0.4

0.3

0.2

0.1

0.0

D i.o

0.9

0.8

0.7

0.6

C,,0 0.5

0.4

0.3

02

0.1

0.0

0 24 48 72

Low hsp27 (n=269)

High hsp27 (n=109)

Low hsp27 (n=269)

High hsp27 (n=209)

96 120

Months

p=O.46p��O.04

0 24 48 72 96 120

Months

Fig. 3 Survival curves (OS and DFS) of ER+/untreated patients as a function of hsp27 expression measured by Western blot (A and B) or IHC (Cand D). The graphs are based on dichotomization of hsp27 at median values.

0 24 48 72

Months

96 120


when present in abundance, would protect tumor cells from a

toxic environment, thereby providing a survival advantage that

would confer a more aggressive clinical course and worse

prognosis for the patient. In a small pilot study using a Western

blot assay, hsp27 overexpression did correlate with a worse DFS

in a group of patients with negative axillary lymph nodes,

despite its association with positive ER, a favorable prognostic

marker (15).

In the present study using a much larger independent

patient sample and two different assay methods, we failed to

confirm our original pilot study. There was agreement between

the Western blot and IHC assays, and with both assays, hsp27

expression correlated, as expected, with ER and PgR positivity.

Neither assay, however, correlated with clinical outcome. Based

on our power analysis, we are confident that we would have

found any clinically significant overall correlation between

hsp27 and outcome in this patient population. This result is in

agreement with several other published studies, although those

typically included patients with positive axillary lymph nodes

who received a variety of adjuvant treatments (16-2 1).

We also divided the cases into four subgroups based on ER

status and adjuvant treatment decision. Analyzing the Western

blot dataset (n = 21 1), hsp27 was correlated with shorter DFS

in ER+/untreated patients. Results from the IHC dataset (n

478) were similar, but subgroup analyses can be problematic,

and the results should be received with caution. However, it is

encouraging that results from two assay methods in only par-

lially overlapping groups of cases were concordant. It has been

known for a long time that hsp27 is estrogen inducible and,

therefore, may be a downstream target in the ER-mediated

growth pathway. Further data also suggest that hsp27 is in-

volved in proliferation processes in breast cancer cells (3, 37).

Also, in our ER-positive subgroup. we found a small but sig-

nificant correlation between hsp27 expression and percentage in

S-phase fraction (data not shown). Adjusting for percentage in S

phase in the prognostic analysis reduces but does not eliminate

the effect of hsp27. Therefore, it is tempting to speculate that

estrogen-induced hsp27 expression could conceivably enhance

the rate of tumor growth and enhance the risk of recurrence in

a patient. The correlation of high hsp27 with shorter DFS but not

OS has been reported previously by Thor et a!. (16) and Love

and King ( I 7). Our hypothesis is that after recurrence, hsp27 is

no longer significant for tumor progression, and its correlation

with and effect on proliferation processes is now overridden by

other mechanisms, such as metastasis or other more aggressive

tumor behaviors. At the moment, there are no data suggesting

that hsp27 is associated with increased tumor metastasis or cell

motility. However, hsp27 is an actin-binding protein and is

associated with actin polymerization (5), and adlin polyrneriza-

lion-associated proteins have been shown to be involved in cell



8. Moretti-Rojas. I.. Fuqua. S. A. W.. Montgomery. R. A. I.. and

McGuire. W. L. A eDNA for the estradiol-regulated 24K protein:


motility (38, 39). We are currently analyzing the melastatic

behavior of breast cancer cells transfected with hsp27 eDNA.

Considering the relatively small sample size of treated

patients, it was difficult to speculate on any influence of drug

sensitivity and/or hsp27 induction by chemotherapy on our

finding in untreated versus treated patients. The direct influence

of adjuvant therapy on the outcome of patients overexpressing

hsp27 could not be evaluated in this study. since the number of

treated lymph node-negative patients with long-term follow up

is very low, and furthermore, the treated patients do not repre-

sent a homogeneous group receiving standardized treatments.

Although the apparent effect on DFS is too weak to be of

practical clinical value, our findings may shed light on the

biological role of hsp27 in breast cancer. These findings may

also help to explain the results published from different groups.

For example. our present studies do not confirm our previous

finding that hsp27 has prognostic power as an independent

marker in lymph node-negative breast cancer in general (15).

However, the patient group analyzed previously differs some-

what from the one analyzed here. In particular, the previous

dataset was smaller but had a greater percentage of untreated

patients with larger tumors and higher S-phase values. The

failure to confirm the prognostic significance of hsp27 in the

overall analyses emphasizes the need to perform validation

studies when characterizing new putative biomarkers.

The data presented here does indicate that hsp27 is not an

independent marker for breast cancer prognosis in axillary lymph

node-negative patients and is, therefore, not a useful tumor marker.

However, it has modest prognostic value in the subgroup of ER+/

untreated patients, which raises new questions about the biological

function(s) of hsp27 in breast cancer cells.

ACKNOWLEDGMENTS

We thank Adrian Lee for critically reading the manuscript and for

many helpful discussions.

REFERENCES

1. Ciocca. D. R.. Oesterreich. S.. Charnness G. C.. McGuire. W. L.. and

Fuqua. S. A. W. Biological and clinical implications of heat shock

protein 27.000 (hsp27): a review. J. NatI. Cancer Inst.. 85: 1558-1570.

1993.

2. Huot, J., Roy, G., Larnbert, H., Chretien. P., and Landry, J. Increasedsurvival after treatment with anticancer agents of Chinese hamster cells

expressing the human Mr 27,()00 heat shock protein. Cancer Res.. 5/.

245-252, 1991.

3. Oesterreich, S.. Weng. C-N.. Qiu, M.. Hilsenbeck, S.. Osborne.

C. K.. and Fuqua. S. A. W. The small heat shock protein hsp27 iscorrelated with growth and drug resistance in htirnan breast cancer celllines. Cancer Res.. 53: 4443-4448. 1993.

4. Miron. T.. Wilchek, M.. and Geiger. B. Characterization of an

inhibitor of adtin polymerization in vinculin-nich fraction of turkey

gizzard smooth muscle. Eur. J. Biochern., 178: 543-553. 1988.

5. Miron, T., Vancornpernolle. K., and Vandekerckhove, J. A 25 kDa

inhibitor of adtin polymerization is a low molecular mass heat shock

protein. J. Cell. Biol., /14. 255-261, 1991.

6. Horwitz. J. Alpha-crystallin can function as a molecular chaperone.

Proc. Nat. Acad. Sci. USA, 89: 10449-10453. 1992.

7. Jakob, U., Gaestel. M., Engel, K., and Buchner, J. Small heat shockproteins are molecular chaperones. J. Biol. Chem.. 268: 1517-1520.

1993.

control of rnRNA levels in MCF-7 cells. Breast Cancer Res. Treat.. 1/:

155-163. 1988.

9. Fuqua, S. A. W.. Blurn-Salingaros. M.. and McGuire. W. L. Induc-

lion of the estrogen regulated “24K” protein by heat shock. Cancer Res..

49: 4126-4129, 1989.

10. Ciocca, D. R.. Adams, D. P.. Edwards. R. J.. Bjercke. R. J.. and

McGuire, W. L. Distribution of an estrogen-regulated protein with a

molecular weight of 24.0(X) in normal and malignant human tissues and

cells. Cancer Res., 43: 1204-1210. 1988.

I I . Adams. D. J.. and McGuire. W. L. Quantitative enzyme-linked

irnniunosorbent assay for the estrogen-regulated M, 24.0(X) protein in

human breast tumors: correlations with estrogen and progesterone re-

ceptors. Cancer Res.. 45: 2445-2449. 1985.

12. Storm. F. K.. Mahvi, D. M., and Gilchrist. K. Hsp-27 has no

diagnostic or prognostic significance in prostate or bladder cancers.

Urology. 42: 379-382. 1993.

I 3. Tetu, B.. Lacasse, B., Bouchard. H-L.. Lagace. R.. Huot. J.. and

Landry. J. Prognostic influence of hsp-27 expression in malignant

fibrous histiocytorna: a clinicopathological and inirnunohistochernical

study. Cancer Res.. 52: 2325-2328. 1992.

14. Ungar. D. R.. Hailat. N.. Strahler. J. R.. Kuick. R. D.. Brodeur.

G. M.. Seeger. R. C.. Reynolds. C. P.. and Hanash. S. M. Hsp27expression in neuroblastorna: correlation with disease stage. J. NatI.

Cancer Inst.. 86: 780-784. 1994.

15. Tandon. A. K.. Clark. G. M.. Charnness, G. C., Fuqua. S. A. W..

Welch. W. J.. Riehl, R. M.. and McGuire. W. L. Heat shock stress

response proteins: biological and clinical significance in breast cancer.

Proc. Am. Soc. Clin. Oncol.. 9: 84. 1990.

16. Thor, A.. Benz, C.. Moore. D. I.. Goldman. E.. Edgerton. S..

Landry, J.. Schwartz. L.. Mayall, B., Hickey. E.. and Weber. L. A. Stress

response protein (srp-27) determination in primary human breast carci-

nomas: clinical. histologic. and prognostic correlations. J. NatI. Cancer

Inst.. 83: 170-178. 1991.

17. Love. S., and King. R. J. B. A 27 kDa heat shock protein that has

anomalous prognostic powers in early and advanced breast cancer. Br. J.

Cancer. 69.� 743-748. 1994.

18. Darmstrup. L.. Andersen. J.. Kufe, D. W.. Hayes. D. F.. and

Skovgaard Poulsen. H. Irnrnunocytochernical determination of the es-

trogen-regulated proteins Mr 24.000. Mr 52.000 and DF3 breast cancer

associated antigen: clinical value in advanced breast cancer and corre-

lation with estrogen receptor. Ann. Oncol.. 3. 71-77. 992.

19. Tetu, B.. Bnisson. J.. Landry. J.. and Huot. J. Prognostic significance

of heat-shock protein-27 in node-positive breast carcinorna: an irnrnun-

histochemical study. Breast Cancer Res. Treat.. 36: 93-97, 1995.

20. Hurlirnann. J.. Gebhard S.. and Gornez. F. Oestrogen receptor.

progesterone receptor. p52. ERDS. H5P27 and cathepsin D in invasive

ductal breast cancer. Histopathology. 23: 239-248. 1993.

2 1 . Seymour. L.. Bezwoda. W. R.. and Meyer. K. Tumor factors pre-

dieting for prognosis in rnetastatic breast cancer: the presence of p24

predicts for response to treatment and duration of survival. Cancer

(Phila.). 66: 2390-2394. 1990.

22. DressIer. I.. (‘i.. Seamer. L. C.. Owens. M. A. Clark. C. M.. and

McGuire. W. L. DNA flow cytornetry and prognostic factors in 1331

frozen breast cancer specimens. Cancer (Phila.). 61: 420-427, 1988.

23. Wenger. C. R.. Beardslee, S., Owens. M. A., Pounds. G.. Oldaker.

T., Vendelay. P.. Pandian. M. R.. Harrington. D.. Clark. G. M.. and

McGuire, W. L. DNA ploidy, S-phase and steroid receptors in more than

127,000 breast cancer patients. Breast Cancer Res. Treat.. 28: 9 -20.

1993.

24. Clark. G. M.. DressIer, L. G.. Owens. M. A.. Pounds. G.. Oldaker.

T., and McGuire. W. L. Prediction of relapse or survival in patients with

node-negative breast cancer by DNA flow cytornetry. N. EngI. J. Med..320: 627-633. 1989.

25. Tandon, A. K.. Clark. G. M.. Chamness. G. C.. Ullrich. A., and

McGuire, W. L. Her-2/neu oncogene protein and prognosis in breast

cancer. J. Clin. Oncol.. 7: 1 120-1 128, 1989.




26. Tandon, A. K., Clark, G. M., Chamness, G. C., and McGuire, W. L.

Cathepsin D and prognosis in breast cancer. N. Engl. J. Med., 322:

297-302, 1990.

27. Adams, D. J., Hajj, H., and Edwards, D. P. Detection of a Mr 24,000

estrogen-regulated protein in human breast cancer by monoclonal anti-

bodies. Cancer Res., 43: 4297-4301, 1983.

28. Allred, D. C., Tandon, A. K., Clark, G. M., Brown, R. W.,

Abbondanzo, S. L., and MeGuire, W. L. Comprehensive evaluation of

prognostic factors by immunocytochemistry on extremely small samples

(50 mg) of “pulverized” breast carcinomas. Breast Cancer Res. Treat.,

16: 182, 1990.

29. Ciocca, D. R., and Bjercke, R. J. Inmiunocytochemical techniques

using monoclonal antibodies. Methods Enzymol., 121: 562-579, 1986.

30. Molina, R., Ciocca, D. R., Tandon, A. K., Allred, D. C., Clark,

G. M., Chamness, G. C., Gullick, W. J., and McGuire, W. L. Expressionof Her-2/neu oncoprotein in human breast cancer: a comparison of

immunohistochemical and Western blot techniques. Anticancer Res.,

12: 1965-1972, 1992.

31. Elledge, R. M., Ciocca, D. R., Langone, G., and McGuire, W. L.

Estrogen receptor, progesterone receptor, and Her-2/neu protein inbreast cancers from pregnant patients. Cancer (Phila.), 71: 2499-2506,

1993.

32. Cox, D. R., and Oakes, D. Analysis of survival data. New York:

Chapman and Hall, 1984.

33. Hilsenbeck, S. G., and Clark, G. M. A practical method of P-value

adjustment. Stat. Med., 15: 102-1 12, 1996.

34. Hilsenbeck, S. G., Clark, G. M., and McGuire, W. L. Why do somany prognostic factors fail to pan out? Breast Cancer Res. Treat., 22:

197-206, 1992.

35. Bielka, H., Hoinkis, G., Oesterreich, S., Stahl, H-J., and

Benndorf, R. Induction of the small stress protein, hsp25, in Ehrlich

ascites carcinoma cells by anticancer drugs. FEBS Lefts., 343: 165-

167, 1994.

36. Dunn, D. K., Whelan, R. D., Hill, B., and King, R. J. Relationship

of hsp27 and oestrogen receptor in hormone sensitive and insensitive

cell lines. J. Steroid Biochern. Mol. Biol., 46: 469-479, 1993.

37. Mairesse, N., Horman. S., Mosselmans, R., and Galand, P. Anti-

sense inhibition of hsp27 production affects growth rate and cytoskel-

eton organization in MCF-7 cells. J. Cell. Biochern., 19B: 100, 1995.

38. Condeelis, J. Life at the leading edge: the formations of cell pro-

trusions. Ann. Rev. Cell Biol., 9: 41 1-444, 1993.

39. Stossel, T. P. On the crawling of animal cells. Science (Washington

DC), 260: 1086-1094, 1993.



1996;2:1199-1206. Clin Cancer Res S Oesterreich, S G Hilsenbeck, D R Ciocca, et al. cancer patients.prognostic marker in axillary lymph node-negative breast The small heat shock protein HSP27 is not an independent

Updated version

http://clincancerres.aacrjournals.org/content/2/7/1199

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/2/7/1199To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



the small heat shock protein hsp27 is not an independent … · vol. 2, 1199-1206, july 1996...

Documents