Immunohistochemical detection of p53 protein in mammary carcinoma: An important new independent indicator of prognosis?

lmmunohistochemical Detection of ~53 Protein in Mammary Carcinoma: An Important New Independent Indicator of Prognosis?

D. M. BARNES, PHD, E. A. DUBLIN, BSc, C. J. FISHER, MRCPATH, D. A. LEVISON, FRCPATH, AND R. R. MILLIS, FRCPATH In an immunohistochemical patients

with a IO-year

with carcinomas

who express

cells (19% of the cases)

tumor

than those survival

(chi-square,

square,

4.93:

P i

tients.

19.68;

P c .02).

.009)

infiltrating behavior available staining overall

interval

p53 protein

lobular

27.97;

of the presence 11.69;

is seen

the effect and grade

(chi-square, is most

and are difficult is second survival.

W.B. Saunders

to subdivide

Cox multivariate only to node

of the protein after relapse

ductal

(chi-

P < .OOl) pa-

in patients

with

carcinomas

(chi-

heterogeneous

clinical

of currently

shows that p53 majority

in significance

HUM PATHOL 24:469-476.

is

(chi-square,

on the basis

analysis status

13.05;

apparent

II infiltrating

of their

prognosis

P < .OOl), overall

in node-negative

P -’ .OOl) that have a rather

markers.

worse

P < .OOl). and survival

and

that patients

in the majority

(chi-square,

marker to ;~tt~~tc‘t real interest il should provide more information than is currentlv available The abnormal ac.c.umulation of the protein encx~ded l,\r the ~53 tumor suppressor. gvne might offer such information. To test this hypothesis MY studied 195 very wrlll-characterized xlcl well-clo~unlented breast cancer cases with followup periods ranging from 10 to 12 years (median followup, IO.01 years). W’e compared cases I.hat expressed varying amounts of ~53 with those that (do not express it at all with regard to (umor skc, hislologic type and grade, and steroid hormone receptor status, .md also compared disease-free interval, overall survival, and posl-relapse survival in both node-neqat~vc~ and -positive patients.

breast cancer

we found

have a considerably

and node-positive

Furthermore,

square,

follow-up

who do not. The effect

seen on disease-free

6.99;

pilot study of 195 primary median

Copyright

of effect

on

1~ 1993 by

Company

MATERIALS

main clinical reasons for measuring biologic. tumor. 11m4wrs: to provide informarion on the likeI!, browse of tile disease and to help with treatment srktion. In addition. there is IIKK~~interest in the effect 01 the presence of oncogent and tumor suppressor ~CIWS on the etiolqq and the progression of man); carcinomas. ~lllr c-r+B-2 protein has been extensively inctbstigated, hot11 ,tt the molec~~lar level and in clinical laboratories using inl~nunohistochemis~i-y. Initial work axsociatiiig amplification of the gene with poor prognosis in breast c;mc~~r l)atients’ was soon followed 1)~ the re;dization that over-expression of the protein encoded b\; tile gene ( oultl hc detectrd imniunohistoclie~ni~ally.“.” Obs~r\~ations that altc‘rations in p53 were a coninion rvent in cancc‘r’ led to hope for similar research with ttle p53 proteill. In contrast to the stable c-&B-2 protwin. wild-1\-pe 1153 is \ery uns~ahle and, therefore, canII( )I he detvc,tv.tl bv imnlullohistocherllistry. FortunateI!,, most mutatlt lx’3 proteins are relatively stable and are amenable to study with recently introduced antibodies lhat recognk pT;3 protein in routinel), processccl his1~~patliologi~ Inatcrial.‘.” New prognostic nlarkers are being clecelopecl every d,lv and it is itnpossible to measure then1 all. For a nt’w -rher~

xc

1~0

AND METHODS

Patients

lmmunohistochemical Staining ~l‘liree-mic ronieter sections \verr (‘lit f rain d representalive paraffin block from each case, air dried overnight. then stained

469

HUMAN PATHOLOGY

Volume 24, No. 5 (May 1993)

TABLE 1. Distribution of Clinical and Pathologic Characteristics

variables. The relationship between positive staining and clinical outcome was determined in univariate analysis by the log rank test using the method of Kaplan and Me&.” The intcrrelationship between the two prognostic groups and other established prognostic factors was determined in a multivariatr analysis using the Cox proportional haLard model. IL’ RESULTS

.Abhr-aviations:

ER. estrogen

receptor;

PR, progesterone

receptor.

using a peroxidase-conjugated streptavidin biotin technique. The sections were dewaxed and rehydrated. Endogenous peroxidase was blocked by washing the sections in a solution of 1% hydrogen peroxide in methanol for 30 minutes. After washing in phosphate-buffered saline (PBS), 0.85% saline buffered to pH 7.2 (Difco I,aboratories, East Molesey. UK), sections were incubated in a 5% normal swine serum/PBS solution for 20 minutes to block nonspecific binding. This solution was drained and polyclonal antibody CMl” was applied at a dilution of 1: 1,500 in PBS for 1 hour at room temperature. Sections were washed in PBS and then incubated with biotinylated swine anti-rabbit immunoglobulin (Dako Ltd, Highwycombe, UK) diluted 1:300 in 15% fetal calf serum/3% normal human serum in PBS for 1 hour, followed by a 15-minute incubation in a peroxiddse-conjugated streptavidin solution (Oncogene Science, Uniondale, NY) diluted 1: 100 in PBS, with intervening PBS washes. The peroxidase reaction was developed with 0.025% diaminobenzidine/O. 1% hydrogen peroxide in PBS to which had been added 1.25 mL of 1% cobalt chloride and 1 mL of 1Yo nickel ammonium sulphate per 100 mL PBS to enhance the staining reactions. Sections were counterstained with a 0.1 YOnuclear fast red solution in 5% aluminum sulphate. Sections of breast tumor known to stain positively for p53 were used as a positive control and were included in each batch of staining. Primary antibody was substituted with PBS in duplicate test sections for negative controls. Scoring

System

Black nuclear staining of the tumor cells was regarded to be positive and was scored semiquantitatively for both the intensity and the proportion of cells staining: intensity was given scores from 0 to 3 and proportion was given scores from 0 to 4 (1% to 25% = 1; 26% to 50% = 2; 51% to 75% = 3; and >75% = 4). The slides were independently scored by two of the authors and any discrepancies were resolved by subsequent consultation. Tumors were classified in three ways: according to (1) the intensity of staining, (2) the proportion of cells staining, and (3) a combination of the two (score 1 plus score 2). Statistical

Analysis

The chi-squared test was used to investigate the significance of the relationship between p53 staining and individual

470

Log rank analysis for disease-free interval, overall survival, and survival after relapse was carried out using each of the three scoring systems. There was no relationship between either the intensity of staining or the combined score and clinical outcome; however, the proportion of tumor cells that were positive for ~53 was significantly related to prognosis (Fig 1). In view of this, all further analyses were confined to the relationship between established factors of prognosis with the proportion of positively stained cells. The fact that some of the patients received adjuvant chemotherapy is unlikely to have influenced the results as those patients were equally divided between the two prognostic groups (P> .2). Table 2 shows the number and proportion of the positively stained cells in the 195 tumors. Thirty-eight of the cases (19%) had positive staining for p53 in more than 75% of the cells (considered “majority stainers”; Fig 2, top), while there was no expression in 35 (18%) of the tumors (considered “nonstainers”). The remaining 122 tumors (63%) had positive staining in a varying proportion of malignant cells (1% to 75%) (considered “nonmajority stainers”; Fig 2, bottom). Staining intensity tended to be homogeneous within individual tumors, but there was marked variation between tumors. There was a variation in the intensity of the ~53 staining in the majority stainers. In 18 cases it was weak, in six it was moderate, and in 14 it was strong. These results emphasize the necessity of having a sensitive method for detecting the p53 protein that is able to identify even the weakly stained cells. There was no association between either tumor size or node status, and the majority, nonmajority, or negative staining cells. There was a weak association between the proportion of stained cells and steroid hormone receptors, with more of the receptor-negative tumors being majority stainers than the receptor-positive tumors (estrogen receptor, P = .07; progesterone receptor, P = .03). There was a highly significant association between the proportion of p53-positive cells and histologic grade of the infiltrating ductal carcinomas, with more of the grade III tumors being majority stainers than the grade I or II tumors (chi-square, 23.20; P < .0002) (Table 3). The significant association remained when the 23 infiltrating lobular carcinomas and the three tumors of other type were included in the analysis (chi-square, 25.58; P < .002). The fact that the number of special tumor types and infiltrating ductal grade I carcinomas was relatively small is probably related to the selection of cases in that sufficient material had to be available for further immunohistochemical analysis. As many of the above tumors are small and also are in high demand for specialized studies, they were not available for this stucly.

~53 AND PROGNOSIS

IN BREAST CANCER

(Barnes et al)

CHI = II 69 P< Ml1

1

4

1

6 TIME

FIGURE 1. Kaplan-Meier plots for (top) relapse disease-free survival, (center) overall survival, and (bottom) post-relapse survival for majority stainers (score 4) compared with plots for nonstainers and nonmajority stainers (scores 0 to 3).

8

10

8

IO

--i--I2

14

(YEARS)

CHI = 19.68 P< 001

1

4

6 TIME WEARS)

--A I:!

14

CHI = 4.934 P = .J263

J 2

4 TIME

471

6 (YEARS)

8

------------(

10

12

Volume 24, No. 5 (May 1993)

HUMAN PATHOLOGY

TABLE 2.

Nonstainers OR8 NorI-majority

Proportion of Tumor Cells Positive for ~53 Protein

stainers

l-2596 ?5-<50% 50475% Majority stainers >75Yo

When the relationship between positive staining for p53 and prognosis was examined in the group as a whole it was found that the 38 patients with majority staining tumors had a significantly worse outcome in terms of disease-free interval (chi-square, 11.69; P < .OOl), overall survival (chi-square, 16.25; P < .OOl), and survival after relapse (chi-square, 4.93; P < .03) than the patients with minority staining or negative staining tumors (Fig 1). The same effect was seen in both node-negative and node-positive subgroups (node negative: chi-square, 6.99 and P < ,009; node positive: chi-syuare, 13.05; P < ,001) (Fig 3; Table 4). Interestingly, when the effect of majority staining was examined within histologic subtypes in grade III infiltrating carcinomas there was no difference in length of survival between the majority stainers and the remaining tumors (chi-square, 0.87; P > .6)(Fig 4, top). However, majority staining had a profound effect on survival in other histologic types (Fig 4, bottom), including infiltrating ductal grade II and lobular carcinomas (chi-square, 25.72; P < ,001) (Table 4). In the Cox multivariate analysis node status had the most significant effect on overall survival (chi-square, 23.48; P < .OOl), followed by p53 majority staining (chisquare, 17.75; P < ,001). The only other factor that remained in the model was histologic type and grade (chi-square, 5.54; P < .Ot'). When all three factors were included in the model, none of the other indicators had any independently significant effect on prognosis. DISCUSSION The p53 nuclear phosphoprotein is expressed in all normal cells and appears to function in cell cycle regulation as a transcription factor. Damage to the ~53 gene resulting in loss of tumor suppressor function is common in human cancer, and a considerable amount of time and efort is now being devoted to understanding the underlying mechanisms. “’ The topic is fascinating, not only for molecular biologists, but also for those involved in the treatment of cancer. Evidence is accumulating that abnormalities in ~53 (allele loss and/o’ mutation) are associated with indicators of poor prognosis.” Luckily for histopathologists, the majority of mutations stabilize the normally labile protein, thus making it amenable for detection by immunohistochemistry.” This is the first report relating the immunohistochemical detection of p53 to prognosis in archival ma472

terial using polyclonal antibody CM- 1. This study also has a longer follow-up (median follow-up, 10.04 years) than previous reports on p53 in mammary carcinoma, all of which used monoclonal antibodies on frozen tissue. The percentage of cases with positive staining found in the frozen tissue ranges from the 27% reported by Davidoff et al”’ who used one antibody (Pab 1801) in 184 cases, the 53% reported by Walker et al” who used five different antibodies in 70 cases, and the 54% reported by Bartek et al’” who used three antibodies in 170 cases. Kecently, Thor et al, also using only Pab 1801 ,I7 found 23% positivity in formalin-fixed tissue. It seems, therefore, that as more antibodies are used there is an increase in the incidence of positive staining, as might be expected since these n~onoclonals are raised against different amino acids and recognize different epitopes. There are several advantages of using antibody CM1. One is that it is a polyclonal antibody that recognizes many different structural forms of the ~53 protein. This is particularly important since there is evidence from several sources”.‘!’ that mutations in different areas of the gene alter the three-dimensional structure of the protein. Another advantage of CM-l is that it can be used in routinely processed, formalin-fixed material without prior digestion. There may be concern about possible loss of antigenicity during the fixation process and we’ have previously emphasized the care that has to be taken over both the process of fixation and the staining methodology, even with CM-l. Indeed, we noted that different fixation procedures do affect the stability of the antigen but, in general, loss of antigenicity in formalin-fixed material does not seem to be a problem with CM-l. This finding is confirmed by the 82% overall level of positivity found in the present study. There is some disagreement over what constitutes positive staining. For example, Davidoff et al” considered that “widespread expression of the protein” represented positive staining, whereas Walker et al”’ regarded any level between 10% and 95% of stained nuclei to be positive. We believe that it is the proportion of positively stained cells that is important. Whatever the chosen cut-off point between positive and negative staining, there is a consensus among investigators that the presence of staining is generally associated with feaIn studies tures indicative of poor prognosis. I~.I5.17.YO-L’:( with an adequate follow-up time positive staining also is associated with reduced disease-free and overall survival. Allred et al”’ have recently published data on 698 patients in whom frozen tissue was stained with a cocktail

TABLE 3. Relationship Between ~53 Staining and Histologic Grade of Infiltrating Ductal Carcinomas

~53 AND PROGNOSIS

IN BREAST CANCER

(Barnes et al)

$.

FIGURE 2. Immunohistochemical staining for p53 protein using antibody CM1 with a peroxidase-conjugated streptavidin biotin technique showing an infitrating ductal carcinoma with majority staining (top) and an in situ ductal carcinoma with nonmajority staining (bottom) (Magnification ~30C )

(l’ab 180 1 and Pal) 240). Positive staining was found in 52% of the cases; this was associated with ;I significant reduction in disease-free interval a( .5 years (I’ = .OO3). In ;I node-uegatice subgroup #oL L'43 patients, positive staining was 2 vein st twig predict< br of recurrence. In a study of 205 cases of nxunnl;~rv carcinoma. Thor et al” found that the 68 disease-frev iuterval positive cases (2: S-% ) had ii shorter of survival (P = .000X) (I’ = .OOR)l ant1 overall leugth af tel. 6 years of’ 1allow-up. staining is of partic.Our finding 1hat p53 niajority ulur significance ill the better-differentiated infiltrating Iobular c;Ircinonlas ductal arc inoinas and in infiltrating is of c onsrderal~l~~ intcresl and Inay provide \xluable

01 two ~nonoc~lonal ;~ntibodies

propostic

illformation

on

rhesc

tumors

thar

is

not

f’roni other sources. The Iack of significance 4x~:inonias is sur11 I infiltrating duc.tal iii the grade prising. We considered that grade I I I 1umors showing no staining may have lost both alleles. hut this subgroup did not differ- ‘in behavior from tun Iors jvith low-level (Ilc,nni;!jot.it~) staining (Fig -1. top). a\ ailable

Minv

new

prognostic

n~arke~-s

at.c

Iwilig

McGuirc” has murnel~al~vi guidelines necessary for the assessnlelIt of One iuiporta~it feature is the calculation quired to be confident ot thv sxnple significmcr of the results. U’C ha\:e uwcl ~IIYY~I~I;III~~~ to calculate. from thr restlIt\ presently

473

;und

c\7tlilattd

;I number of these markers. of the size of thr sratistical the method of reported here,

Volume 24, No. 5 (May 1993)

HUMAN PATHOLOGY

TABLE 4. Relationship Between a High Proportion of p53-Positive Tumor Cells and Overall Survival in Different Subgroups

the numbers of patients required in future p53 studies. Pilot studies sometimes show differences greater than are subsequently observed when hypotheses are later tested in larger studies. We therefore looked at the confidence intervals on the poor survival rates observed in the majority stainers. The upper 90% confidence interval for this group was 49%. Since the survival of the remaining patients at 10 years was 69%, it is possible that the true difference may be 20% at 10 years, rather than the 35% actually observed in this study. To demonstrate a 20% survival difference with 90% power would require the entry of approximately 500 patients (Freedman’“). Obviously, more patients are necessary for subgroup analysis, but the effect of majority staining appears to be similar in the node-positive and node-negative patients. Any immunohistochemical method of detecting p53 protein must be viewed with care because it cannot be assumed that positive staining is indicative of mutation.“” Among the mechanisms that have been shown to stabilize the p53 protein and to allow it to accumulate is the increase in wild-type protein that occurs in an at-

tempt to stop DNA-damaged cells from replicating.‘7 Alternatively, p53 may be complexed to other proteins, such as that encoded by the MDM2 gene.“x This gene has been found to be amplified in some sarcomas’!‘; a similar amplification could occur in mammary carcinomas leading to the formation of a stable MDM2-~53

CHI = 13.05 P < .OOl

1

220

#I

4N=18

1

I

2

4

6

8

10

12

14

TIME CIEARS)

FIGURE 3. Kaplan-Meier plots for overall survival for (top) node-positive and (bottom) node-negative subgroups for majority stainers (score 4) compared with nonstainers and nonmajority stainers (scores 0 to 3).

O-3 N= 83

I 2

4

6 TIME (YEARS)

8

10

12

474

14

~53 AND PROGNOSIS

IN BREAST CANCER

(Barnes et al)

4N=24

CHII= 8748 P= 6451

J

: 2

4

6 TIME WARS)

8

10

---t-------(

12

14

FIGURE 4. Kaplan-Meier plots for overall survival for (top) grade Ill infiltrating ductal carcinomas and (bottom) grade II infiltrating ductal and infiltrating lobular carcinomas for majority stainers (score 4) compared with nonstainers and nonmajority stainers (scores 0 to .3).

t 1

4N=13 C-3 N= 101

r-m

1

h

b TlhiE (YEARS)

REFERENCES

complex. (:onversely, some mutations, far from stahiking the protein, may hasten its disappearance from the cell. Flrl.t~lerlnore.‘if both alleles are lost no protein will be produced at all. In spite of these provisos the present study shows that patients with tumors in which the tnajorir) of cells express p53 protein have a significantly worse outcome than other patients with a similar stage of disease. Although this is only a pilot study using a small number of’ patients. the results are encouraging and, hopefully. will stimulate the additional research necessary before the measurement of p53 can fulfill the evaluation guidelines for prognostic factors that Mc(;uire so elegantly stated in a recent editorial.“’ .~~firaoro/rcf~~~~~,t~~. The authors are extremely grateful to 1’1olesso~- David I.ane, from the CRC 1,aboratories at the Uni\c.rsitv of Dundre. Dundee. UK. for supplying the CM1 antihodv. ‘I‘he authors also wish to thank the medical and surgical staff’ of the I(:RF (:linical Oncology Unit for their careful follow-up and assessment of the patients, as well as Paul Smith and Walttxr (;rrgorT for analysis of the data and statistical advie c

475

s

io

__I ;2

14

HUMAN PATHOLOGY

Volume 24, No. 5 (May 1993)

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476

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