and Transforming Growth Factor 1 Protein Expression ... · encodes a cyclin-dependent kinase inhibitor that is transcriptionally activated by the p53 tumor suppres-sor gene, transforming

Vol. 4, /499-/506, June /998 Clinical Cancer Research 1499

�21wa��1 and Transforming Growth Factor �1 Protein Expression

Correlate with Survival in Non-Small Cell Lung Cancer

William P. Bennett,’ Waflk S. El-Deiry,

Walter L. Rush, Donald G. Guinee, Jr.,

Andrew N. Freedman, Neil E. Caporaso,

Judith A. Welsh, Raymond T. Jones,

Andrew Borkowski, William D. Travis,

Marian V. Fleming, Victor Trastek,

Peter C. Pairolero, Henry D. Tazelaar,

David Midthun, James R. Jett, Lance A. Liotta,

and Curtis C. Harris2

National Cancer Institute, Bethesda, Maryland 20892 [W. P. B..

A. N. F., N. E. C., J. A. W., L. A. L., C. C. HI: University of

Pennsylvania. Philadelphia, Pennsylvania 19104 [W. S. E-D.]:

University of Utah Medical Center, Salt Lake City, Utah 84132[D. G. G.]; University of Maryland [R. T. J.] and Baltimore VA

Medical Center [A. B.l, Baltimore, Maryland 21201: Armed Forces

Institute of Pathology. Washington. D. C. 20306 [W. L. R.. W. D. T..

M. V. F.]; and Mayo Clinic, Rochester, Minnesota 55905 [V. 1..

P. C. P., H. D. T.. D. M.. J. R. J.l

ABSTRACT

� encodes a cyclin-dependent kinase inhibitor

that is transcriptionally activated by the p53 tumor suppres-sor gene, transforming growth factor �1 (TGF-�1), AP2,

and other pathways. Because p21wafl/ciPl, p53, and TGF-�11

all regulate apoptosis and the cell cycle, we tested the hy-pothesis that their relative protein levels would correlate

with biological features including the survival of non-smallcell lung cancer (NSCLC) patients. We conducted an immu-

nohistochemicab analysis of p2lw�i� and TGF-�1 and

identified four patient groups with distinct survival out-

comes. Concordant p2l��ktP) and TGF-�11 expression (i.e.,

either high p21wafl/ciPt and high TGF-�1 expression or low

p2lwf��)) and bow TGF-�11 expression) predicted 70%disease-free survival at 2000 days of follow-up. Discordant

p2lw�il and TGF-�31 expression (i.e., either high

� and low TGF-�11 expression or low �

and high TGF-f31 expression) predicted 35% disease-free

survival (P = 0.0003; log-rank test). These survival relation-

ships were not attributable to differences in grade, stage, or

Received 9/26/97; revised 3/23/98: accepted 3/23/98.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with I 8 U.S.C. Section 1734 solely to

indicate this fact.

I Current address: City of Hope National Medical Center, Duarte, CA

91010.

2 To whom requests for reprints should be addressed, at Laboratory of

Human Carcinogenesis, National Cancer Institute, Building 37, Room

2COl, 37 Convent Drive MSC 4255. Bethesda, MD 20892-4255. Phone:

(301) 496-2048: Fax: (301) 496-0497: E-mail: [email protected].

p53 status. Although current models do not fully explain

these complex interactions, most of these data fit a paradigm

whereby TGF-fH regulation determines NSCLC survival.

In addition to the survival correlation, we found that highp2l��d1�� protein expression correlated with high tumorgrade (P = 0.014). There is little evidence that p2lw/��

protein levels accurately predict p53 mutation status in

NSCLC; specifically, 20 of 48 (42%) tumors with p53 mu-

tations contained high levels of p2lwa�t� protein. These

findings indicate that �21wafl/ciPI immunohistochemical

analysis may provide useful information concerning the

biological properties of NSCLC.

INTRODUCTION

p21��k1P1 is a CDK3 inhibitor that is transcriptionalby

activated by the p53 tumor suppressor gene ( 1 . 2), TGF-�3 1 (3,

4), AP2 (5), and other pathways (6, 7). Additional p53-inde-

pendent regulators include growth factors such as platelet-de-

rived growth factor and fibrobbast growth factor: cytokines

including granulocyte colony-stimulating factor, interleukin 6.

and IFN-’y; serum (6, 8, 9), and the model mutagen, 254 nm UV

bight (10). � levels fluctuate with cell cycle progres-

sion (1 1), probably due to proteasome-dependent regulation

(12), and there is evidence that posttranslational modification,

along with the ratio of p2lwaf�1Pt:eyclin�CDK protein levels,

may regulate its activity (13). �21wafI/ci�1 has been linked to

terminal differentiation (14-16), senescence (17, 18), and inhi-

bition of apoptosis ( 19, 20). In vitro studies demonstrate tumor

suppressor activity that may be useful for gene therapy (21).

Although p53-dependent induction of �21waf1/ciPI causes cell

cycle arrest after DNA damage (2, 22, 23), and p53-independent

induction might inhibit cell division during some differentiation

programs (14. 16), it is not clear why p2l’�ahI�1P’ would be

induced by growth factors, serum, or tumor promoters.

TGF-�3l inhibits the growth of normal epithelial cells. and

resistance to TGF-� 1 is thought to be a feature of the common

cancers (reviewed in Refs. 24 and 25). Latent TGF-�3 1 is pro-

teolytically activated to a form that binds a surface membrane

serine/threonine kinase receptor to initiate a signal transduction

pathway mediated by the Smad proteins (26, 27). TGF431

arrests the cell cycle by transcriptionally activating a series of

CDK inhibitors including p21” (3, 4, 28), p/S. and

p275’� (reviewed in Ref. 29) and by repressing the expression

of CDK tyrosine phosphatase Cdc25A that leads to tyrosine

phosphorylation and inactivation of CDK4/6 (30). Because

TGF-�3 1 regulates p21””�”, and both modulate apoptosis and

3 The abbreviations used are: CDK, cyclin-dependent kinase: TGF-�3 I.

transforming growth factor � 1 : ADCA, adenocarcinorna; SCCA, squa-

mous cell carcinoma: LCC, large cell carcinoma; IHC. immunohisto-

chemistry: NSCLC, non-small cell lung cancer.

Research. on April 18, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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1500 �21atfh/c�1 and TGF-�3I Predict Survival in NSCLC

4 M. H. Barcelbos-Hoff, personal communication.

cell cycle progression (31-34), we tested the hypothesis that

their protein levels may modulate biological features such as the

survival of NSCLC patients.

MATERIALS AND METHODS

Study Population. Matched primary NSCLC and normal

tissues were obtained from 106 consecutive, surgically treated

patients at the Mayo Clinic (Rochester, MN). These samples were

collected between 1991 and 1992. and survival data of at least S

years are available for all patients. Histological subtypes included

29 SCCAs, 55 ADCAs, and 22 LCCs. A full epidemiological

profile including demographics, family history. occupational expo-

sure, and medical history was available on each patient (35, 36).

The study population consisted of 63 males and 43 females. Be-

cause the study population consisted of surgically treated patients,

the majority of the tumors were in the stage I and stage II catego-

ries. Details of p53 analyses and clinical and occupational correla-

tions have been published (35, 36).

p2lwaf�� IHC and Evaluation Criteria. Formalin-

fixed, paraffin-embedded tissue sections from 106 NSCLC tumors

were stained for p2lwattk1� protein using the EAIO monocbonal

antibody (Oncogene Science, Inc., Uniondale, NY) at a working

dilution of 1:50. Antigen retrieval was performed using citrate

buffer (Biogenex, San Ramon, CA) and a ceramic pressure cooker

heated in a microwave for 30 mm. Immunohistoehemicab staining

was performed on a Ventana automated immunostainer (Ventana

Corp.. Tucson, AZ) using an immunoperoxidase detection system.

Each batch of stains (i.e., up to 40 slides) included a positive

control lung tumor known to overexpress p2lwa��15� and a nega-

tive control. which was the same lung tumor stained with buffer

instead of primary antibody.

All � slides were evaluated by three pathologists

(R. T. J.. A. B., and W. P. B.) for p2lW�hIkh1� protein aceumu-

lation; differences in initial evaluation were resolved by group

review at a multiheaded microscope. The following criteria were

applied to assess intensity, distribution, and pattern. For inten-

sity, absent staining was scored as 0, equivocal staining was

scored as I . clearly positive staining was scored as 2, and strong

positive staining comparable to the positive control was scored

as 3. For distribution, absent staining was scored as 0, staining

of <10% oftumor cells was scored as 1, staining of 10-50% of

tumor cells was scored as 2, staining of 5 1-90% of tumor cells

was scored as 3, and staining of >90% of tumor cells was

scored as 4. For pattern, absent staining was scored as 0,

sporadic or occasional staining was scored as 1, focal or clus-

tered staining was scored as 2, and diffuse or widespread stain-

ing was scored as 3. The scores for intensity, distribution, and

pattern were summed and correlated with survival as well as

other clinical and laboratory data.

TGF-�H IHC and Evaluation Criteria. The TGF-�3l

antibody used in this study (AB-lOl-NA polyclonal antibody;

R&D Systems, Inc., Minneapolis, MN) was raised against re-

combinant human TGF43I and binds both the active and latent

forms of TGF-�3l .� The primary antiserum was applied without

antigen retrieval at a working dilution of I :80, incubated over-

night at 4#{176}C,and then detected by the Ventana automated

immunostainer using an immunoperoxidase detection system.

Each batch of stains (i.e., up to 40 slides) included a positive

control (nonpigmented, nonneoplastic human skin) and a nega-

tive control (the same nonpigmented, nonneoplastie human skin

stained with buffer instead of primary antibody). The slides

were reviewed at a multiheaded microscope by two pathologists

(W. L. R. and W. D. T.) who compared the cytoplasmic staining

of tumor cells to the eonstitutive immunoreactivity of alveolar

tissue. Tumors with increased staining relative to nonneoplastie

alveolar tissue were classified as overexpressers, whereas those

demonstrating immunoreactivity at or below the baseline were

considered to lack overexpression.

Statistical Analysis. The immunohistochemical data

were correlated with survival, clinical, epidemiological, and

laboratory data using the SPSS statistical program version 7.5

for Windows (Chicago, IL). A full epidemiological profile in-

eluding demographics, family history, occupational exposure,

and medical history was available on each patient (35, 36). A

total of 102 of the 106 cases had a detailed profile of smoking

history. Because this study is an ongoing research project, data

from other genetic markers and cell cycle proteins were avail-

able for analysis including p53 (35) and K-ras mutations (77);

allelic deletion of the FlIT tumor suppressor gene (77); the

expression of cyclin Dl (77), e-erbB-2 (35), and Bax/Bcl-2

(37); and genetic polymorphisms of CyplAl, Cyp2EI, and

GSTMJ (38).

RESULTS

p2lwdhI� Immunohistochemical Analysis of Tumors.

The p2 1wafl/cipi immunohistoehemical staining pattern was ex-

elusively nuclear with a typically dense, granular appearance

(Fig. IA). The �21wafI/cIP1 immunohistoehemical results are

tabulated by histology, gender, and staining category in Table 1.

Overall, 23 of 106 (22%) tumors were negative, 5 of 106 (4%)

tumors were slightly stained, 38 of 106 (36%) tumors were

moderately stained, and 40 of 106 (38%) tumors were markedly

stained; there were similar distributions by gender and histol-

ogy. There was no clear association between p21wafllc*P1 IHC

and mutation of p53 or K-ras or between p2l�vaftk1�)t IHC and

p53 IHC.

TGF-�1 Immunohistochemical Analysis of Tumors.The TGF-�3 1 immunohistoehemical staining pattern was typi-

cally diffuse cytoplasmie staining of tumor cells and occasional

staining of the acellular, extracellular matrix near tumor deposits

(Fig. 1B). The TGF-�3l immunohistochemical results are tabu-

bated by histology, gender, and staining category in Table 2.

Overall, 61 of 106 (58%) tumors were negative, and 45 of 106

(42%) tumors were positive (i.e. , overexpressed); there were

similar distributions by gender and histology. There were no

clear associations between TGF-�3l IHC and mutation ofp53 or

K-ras or between TGF-�3l IHC and p53 IHC.

� and TGF-fil Protein Expression PredictsSurvival. We first correlated tertiles of p2 1wafl/cipi protein

expression with survival and found a nonsignificant trend of

reduced survival with increasing amounts of p2 1 wafl/cipi protein

(P = 0.09, log-rank test). We also investigated combinations of

p21wa�k1P1 and p53 mutation or protein accumulation, but no



‘A

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s’.-

, .v�Is.

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: � �

S

(44

- � � .. -� - Table 1 p2l��oJk�)I immunohistochemical analysis by histology.

,p #{149}� � and gender

p2l#{176}�”�”IHC”

I,

�‘b

4

.,�

0

a

5�

I.

a

p

Tumor feature Negative Slight Moderate Marked Total

I.

....

4

“i� #{149}�

S4

IIb#{149}.�

‘�

‘�‘

�. q;q>’

...

�

‘�A� .

#{149}. .#{149}��Auj.’ �

a . #{149}1_tb �

r �

S 2 7 12 26

8 0 13 8 29

2 3 9 8 22

2 0 3 2 7

4 0 4 7 15

2 (1 2 3 7

23 5 38 40 106

6 3 9 9 27

6 0 4 Il 21

2 2 6 10 20

5 0 10 3 18

19 5 29 33 86

8 3 16 II 38

2 0 3 6 II

I I 6 8 16

lb I 13 16 41

22 5 38 41 106

-.

. . . �,r-

. a-’

“ The categories negative. slight. moderate, and marked are sum-

mary scores (i.e., intensity + distribution + pattern) ofO, 1-3. 4-6, and

, 7-10, respectively.

I, A total of 86 cases was selected for tabulation by excluding cases

� �‘ with multicentric tumors or incomplete mutation analysis.

. .�. �:.4 ‘a

. .�,‘a- 0.0003. log-rank test) and were not attributable to differences in

Fig. / A, irnrnunohistochernical analysis of p2b’��hh/d’P’ in NSCLC. grade. stage, or p.53 mutation status.

Dense accumulations of �21wafI/ci�I protein occurred in the large ma- Correlation of p21W��PI and p53 Protein Levels withjority of tumor nuclei in this field (X400 magnification). B. imrnuno- p53 Mutation Status. Among 48 tumors with p53 mutation,histochemical analysis of TGF-�l protein in NSCLC. Heavy cytoplas- 20 (42%) had high levels of �21wu�k��1 protein, and among 38mie deposits ofTGF-�3l protein occurred in all tumor cells in the middle tumors with wild-type p.53, 9 (24%) had little or no �of the microscopic field ( X480 magnification).

protein. Among 41 tumors with marked p53 protein expression,

16 (39%) had marked p2lwa��1�)I protein. and in 38 tumors with

negative p53 protein expression. 8 (2 1%) lacked p2 1 waS SkIPS

protein expression. These data deviate substantially from theplausible survival correlations were found. Specifically, subsets simple model linking p2b�”'”�1 protein expression to func-

with wild-type p53 sequences containing low, medium, or high tional, wild-type p53 expression. We also tested the hypothesis

levels of p2lwaftkht� protein did not have characteristic survival that a ratio of p2 1 ��‘�f1k1�)1 p53 proteins determined by IHC could

outcomes; similar observations were made from tumors contain- predict the functional status of the p53 gene. This hypothesis

ing p53 mutations. leads to a model illustrated by two examples: (a) a tumor

The highest and lowest tertiles 0fp21�tk1��1 and TGF-�3l containing high levels of p21wa�k1�)1 protein and low levels of

protein accumulations produced significant survival correlations � protein may have a functional (i.e. . wild-type) p53 gene,

(Fig. 2). Concordant � and TGF-�3l expression (i.e., because low levels of wild-type p53 protein may produce sig-

either high � and high TGF431 expression or low nificant amounts of p2 1 ��1kp1 protein by transcriptional acti-

�21waf1kt�1 and low TGF431 expression) predicted 70% dis- vation; and (b) a tumor containing low levels of p2l’�”t1”

ease-free survival up to 2000 days posttreatrnent. Discordant protein and high levels of p53 protein may have a dysfunctional

p2P��afh/d1Pt and TGF-�3l expression (i.e., either high � (i.e., mutated) p53 gene. because mutant p53 protein typically

and low TGF-�3l expression or low p2lwafl/CIPI and high accumulates to high levels but is unable to generate significant

TGF-�3l expression) predicted 35% disease-free survival. The amounts of p21 W�Ifl/CI�l protein by transcriptional activation.

differences between the concordant (ii = 44 patients) and dis- Using these models, we compared the protein levels in 86 lung

cordant (n = 45 patients) groups were highly significant (P = cancers with known p53 mutation status (35). We found that

Clinical Cancer Research 1501

Histology

ADCAMF

SCCAMF

LCCM

� #{149}1 FTotal

p53 rnutation”

Yes

M

FNo

MF

Total

p53 IHCNegative

Slight

Moderate

Marked

Total



p = 0.0003

-� I

� -�

100

80

60

40

A

B

20

0 1000

>

U)

>

C,)

2000 3000

100

80

60

40

20

I�\ p=0.0003

- **

“ A total of 93 cases were selected for tabulation by excluding caseswith multicentric tumors or incomplete mutation analysis.

ratios of p2 1 waS I/cap1 p53 protein correctly predicted 63% of p53

mutations and 59% of wild-type tumors. We compared these

results to those with p53 IHC alone: 70% of tumors with intense

and widespread p53 protein accumulation had missense muta-

tions; and 62% of tumors without p53 protein accumulation

contained the wild-type sequence. We concluded that ratios of

p53:p2 I � protein do not correlate well with p53 mutation

status in NSCLC.

Correlations with Tumor Grade and Stage. The high-

est tertibe ofp2r��uk1P protein correlated with high grade (P =

0.014), and there was a nonsignificant trend toward reduced

survival in high-grade (i.e. , high nuelear:eytoplasmie ratio) tu-

mors (P = 0. 17). p2 1 wati/cipl staining does not correlate with

stage in these NSCLCs. TGF-�3l immunoreactivity did not

correlate with stage or grade.

DISCUSSION

p2lw��i� and TGF-�1 Protein Expression Predict

Survival. Although neither p21wafIk1Pt nor TGF-�3l protein

levels predicted survival individually, combinations of the high-

est and lowest tertiles of protein expression identified four

subgroups with distinct outcomes (Fig. 14). Two groups had

relatively good survival: (a) those with low TGF-f31 and low

p2l�va�/d1�) expression; and (b) those with high TGF-�3l and

high p21w�f�1��5 expression. The two remaining groups (those

with low TGF-�3l and high p21wah5kut� expression and those

with high TGF431 and low p2lwaw�I expression) had poor

I 000 2000

1502 p2lwoh/c1� and TGF-�3b Predict Survival in NSCLC

Table 2 TGF-�3l irnrnunohistochemical analysis by histology.p53 status, and gender

Tumor feature

TGF-�3l IHC

Negative Positive Total

Histology

ADCAM 16 10 26

F 18 II 29

SCCAM 10 12 22

F 3 4 7LCC

M 10 5 15F 4 3 7

Total 61 45 106

p53 mutation”

Yes

M 18 9 27F 13 5 18

No

M 15 10 25F II 12 23

Total 57 36 93

p53 IHCPositive

M 13 14 27

F 11 7 18Negative

M 18 17 35F 13 12 25

Total 55 50 105

Disease Free Survival (Days)

Disease Free Survival (days)

Fig. 2 p2lw4f1khI� expression and TGF-�3l protein expression predict

survival in NSCLC. A, four groups of patients were defined by the

highest and lowest tertiles ofp2lwa�/d1�I and TGF-�3l protein expression

determined by IHC. Two groups did well with extended periods of

>60% survival: patients with low TGF-�l and low p2lwath�1� expres-

sion (dashed line; a = 30) did slightly better than those with high

TGF431 and high p2lwafl/cIPI expression (solid line: pi = 15). Two other

groups had significantly worse survival (P = 0.0003, log-rank test):

tumors with low TGF-�3l and high �21waf1��1 expression (double

dashed line; n = 24) and those with high TGF-�3l and low p2l�ao1k�)1

expression (double solid line; “ = 26). + and �, censored. B, to simplify

the data and illustrate a mechanistic framework, we consolidated the

four groups into two groups; tumors with concordant (i.e. , both high or

both low) protein levels (dashed line: ii = 45) had significantly better

survival than those with discordant (i.e. , one high and one low) protein

levels (solid line; ii 50) (P = 0.0003, log-rank test). These survival

correlations were not attributable to differences in grade, stage. or p53

mutation status. �, censored.




survival. To simplify these data and illustrate a mechanistic

framework, we can consolidate these four groups into two

groups; those with concordant (i.e., both high or both low)

protein levels had significantly better survival than those with

discordant (i.e., one high and one low) protein levels (Fig. 2B;

P = 0.0003, log-rank test). These survival correlations were not

attributable to differences in grade, stage, or p53 mutation

status.

These complex data fit a model (39) which posits latent

TGF-�3l activation as a critical barrier during preneoplasia that,

once surmounted, leads to selective pressure for TGF-�3l resist-

ance. This paradigm assumes that activation of TGF-�3l is

highly restricted in normal tissue but can accompany certain

tissue processes and tumor/stromal interactions. If widespread

activation does not occur, then the low-low expression pattern

results, but if activation occurs late in tumor progression, then

the high-high expression pattern develops. We consider both

conditions to be favorable, because they indicate that TGF-�3l

activation per se is still regulated. Independent experiments

support this model, because it is known that TGF-�3l induces

� (4 40) and other CDK inhibitors, p15 and p27”

(41), to produce cell cycle arrest or apoptosis. Furthermore, the

high-high phenotype corresponds to TGF-�3l-sensitive breast,

ovarian, and gastric cancer cell lines that up-regulate p21”�#{176}1”

expression in response to TGF-�3l (42-44).

This model also explains half of the discordant group (Fig.

2B): the tumors with high TGF-�3l and low �21waf1�1 expres-

sion fit a scenario in which TGF-�3l activation occurs early in

progression, and the tumor develops resistance such that

� is not up-regulated to induce cell cycle arrest or

apoptosis. This phenotype may illustrate the deleterious combi-

nation of tumor cell TGF-�3l resistance and the nonepithelial

consequences of unbridled TGF-�3l activity: (a) immune sup-

pression; (b) angiogenesis; (c) extracellular matrix deposition;

and (d) protease regulation. Furthermore, this pattern fits the

breast, ovarian, and gastric cancer cell lines that are resistant to

TGF-�3l inhibition and show little or no � expression

when exposed to TGF-�3l (42-44). In addition, these tumors

might be growth-stimulated by TGF431, as observed in some

metastatic colon cancers (45, 46). The tumors with low TGF431

and high p21wa�c� expression do not fit easily into the current

model, although new insights into the TGF-�3l signaling path-

way may accommodate this group. For example, analyses of the

p15 and p27”'’ components of the TGF-�3l pathway might

uncover a plausible link to this subset. Finally, these tumors

might be connected by regulators of the apoptosis pathway,

because p21wafl/cIPI and TGF-�3l modulate and induce apopto-

sis, respectively (20, 34, 47, 48).

Literature Review of p2lw�A�u1� Protein Expressionand Survival. Two series of NSCLCs, totaling 197 patients,

have found evidence for improved survival with increased

�21waf1�� expression (78, 79); larger series plus uniform cri-

teria and methodologies are needed to determine the correlation

between �21waf1/ci�I and survival in NSCLC. Two groups have

correlated �21waf1/c*�1 overexpression measured by IHC with

adverse outcome in breast cancer. An analysis of a series of 261

patients linked p2 1 wafl/cipi overexpression with large tumor

size, positive nodal status, high histological grade, high mitotic

count, and short disease-free survival (49). Among patients

treated with chemotherapy, tumors with high levels of both

p2lwa��1�)I and p53 proteins had significantly greater overall

survival compared to p21 -ip53 + tumors (P = 0.00001); rnul-

tivariate analysis showed that the p21 -/p53+ phenotype was an

independent predictor of survival (49). Using a second series of

91 breast carcinomas, it was found that high �21wafI/ciPI expres-

sion alone correlated with short relapse-free survival (P =

0.003; Ref. 50). In contrast, a cohort of 40 prostate cancer

patients found no correlation between p2lw��ftk1�)t staining and

clinical grade, stage, or tumor progression (5 1 ). An analysis of

one series of colon cancers found an inverse relation between

p2lwaf�� and stage, suggesting a down-regulation of

‘a”’ during progression (52).

p53 and Survival in NSCLC. With more than S years

of follow-up and nearly 50% of the cohort surviving, there is

no correlation between p53 mutation or protein accumulation

and survival in our series of predominantly stage I and II

NSCLCs. Although p53 status generally predicts survival in

breast cancer (53), there is no clear correlation for NSCLC,

because p53 mutation or protein accumulation has been

linked to better (54-58), worse (59-67), or neutral (68-71)

outcomes.

p2lw��/c�� Expression and Correlations with p53Functional Status. The concept that protein levels of down-

stream effectors ofp53 reflect p53 functional status is appealing

but unproven to date. In addition to our data, the analysis of a

series of 21 pancreatic tumors found that immunocytochemical

analysis 0f�2lwafI/cIP1 protein did not reflect the mutation status

of p53 as determined by sequence analysis (72). An analysis of

a series of NSCLCs found high levels of the � iran-

script in 1 1 of 14 tumors showing both p53 gene mutation and

p53 allelic loss (73) and did not find a clear relationship between

the levels of p21”�” message and p.53 mutations. An im-

munohistochemical analysis of 91 primary breast cancers found

no association between the expression of p2lwafh/��l and p53

(50).

Conversely, data from colon and ovarian tumors do support

a correlation between p2J’”�” expression and p53 func-

tional status. For example, an analysis of a series of 23

primary epithelial ovarian cancers found that normal levels of

p21”-”1” RNA were seen in 4 of 7 (57%) cancers with

wild-type PS3, whereas 14 of 16 (88%) cancers with mutant p53

had reduced �21wafI/ciPI expression (P < 0.05). Elbendary et a!.

(74) concluded that mutation of the p.53 gene in ovarian cancers

is usually associated with decreased � expression.

Analysis of a series of 16 colon tumors found that p2J’�”-””�”

mRNA expression in tumors with p53 mutation was about 60%

that of cases lacking p53 mutation (75). Another series of 40

colon cancers found that all 5 tumors with decreased p2lwafl/clPI

expression had p53 mutations, whereas normal or increased

p2lwaf�� expression was almost evenly divided between

those with mutant and wild-type p53 status (52).

Conclusions. Immunochemical analysis of p21 wafi/cipl

and TGF-�31 identifies cohorts with very different survivals, and

we propose a functional model that may explain most of these

observations. It is notable that high levels of p2lwaul/cIPI, PS3,

and cyclin Dl proteins predict short survival, whereas low levels



l5O4p2b�’��h1k1P1 and TGF-�31 Predict Survival in NSCLC

predict long survival.5 The correlation of high p2lwafl/cIPI pro-

tein with high-grade (i.e., high nuelear:eytoplasmie ratio) is

consistent with reports linking p2lwa�1�)I overexpression to

giant cell formation in human cancer cell lines (47, 76). Ratios

of p2lwafl/cIPl:ps3 protein levels do not correlate well with p53

mutation status in NSCLC. These and other ongoing studies

show that the differential expression of growth factors, CDK

inhibitors, and tumor suppressor genes correlates with biological

features including survival.

ACKNOWLEDGMENTS

We thank Drs. Bert Vogelstein (Johns Hopkins University. Balti-

more, MD) and Thomas V. Colby (Mayo Clinic) for seminal contribu-

tions to the early phases of this project, and we thank Dr. Mary Helen

Barcellos-Hoff (Berkeley National Laboratory, Berkeley, CA) for help-

ful discussions and communications. We also thank Ricardo V. Drey-

fuss (NIH, Bethesda, MD) for expert photornicrography.

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1998;4:1499-1506. Clin Cancer Res W P Bennett, W S el-Deiry, W L Rush, et al.

cancer.expression correlate with survival in non-small cell lung p21waf1/cip1 and transforming growth factor beta 1 protein

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and Transforming Growth Factor 1 Protein Expression ... · encodes a cyclin-dependent kinase inhibitor that is transcriptionally activated by the p53 tumor suppres-sor gene, transforming