[CANCER RESEARCH56, 3415-3420. August 1. 19961
Advances in Brief
Rb and
@16@4a Expression
Robert A. Kratzke,2
Gloria
in Resected
Todd M. Greatens,
A. Niehans,
and Joseph
Non-Small
Cell Lung Tumors'
Jeffrey B. Rubins, Michael A. Maddaus,
Dennis E. Niewoehner,
Geradts
Department of Medicine, Sections of Hematology/Oncology
[R. A. K.J and Pulmonary Medicine IT. M. G.. J. B. R., D. E. N.], and Departments of Pathology 1G. A. N.J and
Surgery (hf. A. Mi, Minneapolis Veterans Affairs Medical Center and the University of Minnesota Medical School, Minneapolis. Minnesota 55417, and Department of Pathology
and Laboratory Medicine, University ofNorth Carolina School ofMedicine, Chapel Hill, North Carolina 27599 fJ. G.J
Abstract
@
Inactivation ofthe cyclin-dependent kinase inhibitor p16@'@@(CDKN2/
MTSJ) is documented in a wide variety ofcancer cell lines and tumors. We
have shown that loss of
protein expression is a common event in
early stage non-small cell lung cancer (NSCLC), correlates with a signif
pRB (7). Phosphorylation
of pRB leads to release
of the cell from
15% of the tumors, whereas 51% possessed aberrant p16@@a protein
Rb-mediated growth inhibition and subsequent entry of the previously
quiescent cell into S-phase (8). In the transformed cell, overexpression
of members of the S-phase family of cyclins (cyclins Dl—D3and E)
can presumably drive the cdk-mediated phosphorylation of the RB
protein, allowing the cell to leave G0-G1 (9, 10). Alternatively, mu
tation or deletion of the Rb gene will result in a permanent removal of
Rb-mediated cell growth inhibition, thus favoring unrestricted divi
expression.
sion
icantly worse survival, and is more common in higher stage disease. One
hundred NSCLC tumors from patients undergoing definitive thoracoto
mies at a single institution were examined for @16@N@
and retinoblas
toma protein
(pith)
expression.
Tumors
Abnormal
with aberrant
pith staining was identified
expression
of pl6―@
in
by immunohis
tochemistry were associated with a significantly worse survival (P = 0.04).
Additionally,
the inverse
correlation
of pRB and
@16IN@ expression
previouslyliotedin lung cancer celllinesand tumors wasconfirmedin this
large cohort of patients, with 65% of the tumors demonstrating
inverse
expression of pRB and p16@'― (P = 0.00019). A statistically significant
increase in aberrant p16@―@
expression, as well as inverse expression of
p16@N@@@
and pRB, was seen with increasing
pathological
stage of disease.
These findings establish the prognostic significance of the absence of
p161@
in resected
NSCLC
and
confirm
the
critical
importance
of
disrupting the pathway of cydlin-dependent kinase-mediated phosphoryl
ation of pRB in the molecular oncogenesis and progression of NSCLC.
Introduction
@
products is evident in view of the known function of the @J6@@NK4@I
gene product as an inhibitor of cdk4-mediated phosphorylation of
of the
cell.
The
importance
of releasing
the pRB-mediated
inhibition of cell growth in the genesis of NSCLC is strongly under
scored by the inverse correlation of expression of wild-type pRB and
the cdk4 inhibitor p16INK4a in lung cancer cell lines (6) and tumors
(11—13).
To establish the incidence of Rb and pJ6IN@@4a
gene product ex
pression in a large cohort of early stage NSCLC, we have examined
a consecutive series of 100 patients undergoing resection of NSCLC
for curative intent at a single institution (MVAMC). Resections were
performed over a period of 4 years and included patients thought to
have clinical stage I, II, or lILA as determined by conventional
preoperative staging. We found that the absence of the @J6m@c4@@
gene
product is a common occurrence in resected lung cancer and corre
lates significantly with the presence of wild-type pRB expression.
Conversely, the less frequent occurrence of absent Rb gene product
expression is usually accompanied by the presence of normal appear
ing (wild-type) p16INK4a protein. In addition, absence of
expression is an indicator of a worse prognosis in patients undergoing
resection for NSCLC and is associated with more advanced patho
logical stage. Finally, adenocarcinomas of the lung have a much lower
incidence of abnormal pl6LNK4aexpression and a higher incidence of
Non-small cell lung cancer (NSCLC)3 will be newly diagnosed in
approximately 177,000 people in the United States in 1996 and will
cause more than 150,000 deaths, making it the single largest cause of
cancer death in this country (1). Although the vast majority of these
cases are associated with the use of tobacco products, the underlying
genetic targets of the carcinogenic effects of tobacco use have only
become known in the last decade (2). Acquired genetic mutations in
NSCLC have been identified in a number of tumor suppressor genes,
including a high frequency of mutations in the Rb, p.53, and pJ6@―4― coexpression of pRB and p16INK4a than other histological
genes. Additional frequent alterations in the expression of myc and ras
NSCLC.
oncogenes have also been associated with the development of NSCLC
(3, 4). Although the rate of acquired aberrations for these molecular
Materials and Methods
subtypes
of
targets vary, the majority of lung cancer tumors and cell lines studied
possess identifiable abnormalities in one or more of these growth and
cell cycle regulatory elements (5).
It has recently been reported that the subset oflung cancer cell lines
that express a normal (wild-type) Rb gene product is likely not to
express the pJ6INK4a gene product (6). The importance of the inverse
correlation in protein expression between the Rb and p16@―4―
gene
Received 5/7/96; accepted 6/14/96.
The costs of publication of this article were defrayed in part by the payment of page
charges.Thisarticlemustthereforebe herebymarkedadvertisementin accordancewith
18 U.S.C. Section 1734 solely to indicate this fact.
I This
work
was
supported
by a Research
Advisory
Group
grant
from
the Department
whom
requests
for
reprints
should
be
addressed,
at
Section
of
Hematology/
Oncology, 1I1E, Minneapolis Veterans Affairs Medical Center, Minneapolis, MN 55417.
Phone: (612) 725-2000, ext. 4135; E-mail: [email protected].
3 The
abbreviations
the MVAMC were examined for diagnosis. One hundred twenty-three of these
cases were determined
to have primary NSCLC,
which was surgically
staged
and resected for cure. Fifteen of these cases had insufficient remaining tissue
for further studies. Of the remaining cases, two were found to have resected
lesions
likely
to be metastatic
disease
rather
than
primary
NSCLC.
Two
additional patients had insufficient follow-up data to determine survival. One
patient underwent two thoracotomies in the period of this study, with similar
histologies, and the second lesion was considered likely to represent recurrent
disease rather than primary NSCLC. In total, 102 were available for evaluation
in this study. Two of the pathology samples did not stain adequately after
repeated attempts and were thought to be degraded. The pathological stages,
of Veterans Affairs (R. A. K.).
2 To
Patient Population and Surgical Samples. The pathologyreportsof pa
tients who underwent thoracotomy between July 1990 and December 1994 at
used are: NSCLC,
non-small
cell
lung
cancer;
cdk,
cyclin-depend
ent kinase; RB, retinoblastoma; MVAMC, Minneapolis Veterans Affairs Medical Center.
histological subtypes, and ages of the remaining 100 cases are listed in Table
1. All of the patients were men. Exposure to cigarette smoke or environmental
or occupational pollutants was not determined. Patient information was avail
able from the MVAMC Tumor Registry and chart review. Survival data were
3415
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Rb AND
patientsMean
Table 1 Clinical characteristics
(7.5)Median
age
@16INK4a
EXPRESSION IN NON-SMALL
of 100 thoracotomy
CELL LUNG TUMORS
of expression of either tumor suppressor gene. If stained nuclei were present in
all areas of the tumor, it was considered normal. If there was absence of
(yr) at time of diagnosis (SD)66.4
surgery49Mean
follow-up(mo)since
nuclear staining in a portion of or an entire tumor section, whereas admixed
(14.9)Histology
follow-up (mo) since surgery (SD)47.4
nonneoplastic
100)Adenocarcinoma47Squamous
of tumors (n
cells did show nuclear reactivity,
the lesion was considered
abnormal.
StatisticalAnalysis.Associations
betweencategoricalvariableswerean
carcinoma42Large
cell
alyzed with @2
analysis. Survival curves were calculated by the Kaplan-Meier
method and compared with the log rank test. Independent survival variables
were studied by Cox regression analysis. Calculations were performed using
SPSS for Windows (release 6.1; SPSS, Inc., Chicago, IL). All P values are
carcinoma5Adenosquamous
cell
carcinoma6Pathological
100)I59II24III16IVI
stage of tumors (n =
two-tailed.
Results
Absence of detectable p1611@4'@
gene product expression is a com
mon occurrence in early stage NSCLC (Table 2). Approximately half
of the tumors examined had absent or abnormal p16@4a expression
the MVAMC. Most of the operations were performed by the same thoracic (Table 2) (Fig. 1). The subset of pl61'@4―-negative tumors is distrib
surgeon (M. M.). All surgical samples were processed for routine diagnostic uted among the four histological subtypes examined, but is less
studies, including H&E staining. The diagnostic material was reviewed by a
commonly seen among adenocarcinomas and large cell carcinomas.
single pathologist (G. N.) and was classified as one of four histological
The majority of the pl6I4&@@negativetumors showed complete loss
subtypes: adenocarcinoma, squamous cell carcinoma, large cell carcinoma, or
of p16INK4a reactivity, whereas admixed nonmalignant cells served as
adenosquamous carcinoma. All samples were subject to routine electron mi
internal positive controls (Figs. 1 and 3). In paraffin-embedded archi
croscopy at the MVAMC to further confirm the histological subtype.
Materials. Murine monoclonal a-RB antibody 3C8 was purchased from val tissues, it has been reported that lymphocytes do not uniformly
QED (San Diego, CA; Ref. 14). Murine monoclonal a-RB antibody PMG stain positive for p16INK4a (15), and this was seen in some of our
3—245 and rabbit polyclonal a@pl6@@4a antiserum were obtained from
samples, although positive elements were easily identified on most
PharMingen (San Diego, CA; Refs. 15 and 16. Nonspecific mouse IgGl or rabbit
sections. Four of the tumors that were reported as negative for
serum,respectively,wasusedas a negativecontrolreagenLThedetectionreaction p16@@4a expression had focal areas of pl6@@4a
@@@1th@1ty
(Fig. 2).
usedthe Vector(Burlingame,CA) Eliteavidin-biotincomplexkit
These tumors had clearly demarcated areas of p16INK4@@@negative
cells
Immunohistochemistry. The immunohistochemical assays for detecting
(with p16―@@-positive stromal elements intermixed). Whether this
pRB and p16INK4ain fixed and paraffin-embedded tissues have been described
represents tumor heterogeneity or is a staining artifact is open to
in detail elsewhere
(14—16). Five-sm
sections were used, which had been
question, but it is unlikely to represent a p16@@4a@positivetumor that
freshly cut or stored at 4°C.Briefly, for detecting pRB, sections from all 100
is failing to stain adequately, given the continued presence of detect
cases were reacted with antibody 3C8 at 1 mg/ml for I h. A subset of cases
were also reacted with PMG 3—245
at 2 mg/mi for 2 h. In both instances, the able p16INK4aprotein in the nonmalignant tissues (Figs. 1 and 3). This
primary incubation followed a 20-mm antigen retrieval step in hot citrate focal positivity is similar to that previously reported in mesothelioma
buffer. Negative control slides were treated with nonspecific mouse IgGl at and other tumors (15, 16). Two of these four tumors were from
equivalent conditions. The staining protocol for pl6tN@@4a
did not include an
patients with subsequent metastatic disease (brain and chest wall).
antigen retrieval step. Sections were reacted with pl61T@'4@'
antiserum (negative
These metastatic lesions were available for analysis and were found to
controls with preimmune
rabbit serum) at a 1:400 dilution at 4°C overnight.
be uniformly negative for pl6INK4aexpression (Fig. 4). In general, the
The Vector Elite avidin-biotin complex kit provided the secondary reagents.
percentage of pl6tNK4@@@negative
tumors increased correspondingly
For color development of both pRB and pl6@4―stains, we used diamino
with
stage
(Table
2).
pl6@4―
was
absent in 41% of stage I tumors,
benzidine with hematoxylin as a counterstain. For scoring the pRB and
63% of stage H tumors, and 75% of stage HI tumors. Although the
p16@4―
staining
patterns
as normal
or abnormal,
we used previously
pub
lished criteria (15, 16). Cytoplasmic reactivity was disregarded, and only number of tumors in each subgroup was small, this trend was sigmf
nuclear staining above any cytoplasmic background was considered evidence
icant by x2 analysis (P = .02391). A similar increase in the incidence
considered
inadequate
if the patient had not been evaluated
as an inpatient or
outpatient at the MVAMC for the previous 6 months, could not be confirmed
as deceased, or could not be contacted by appropriate patient representatives of
Table 2 pRB and @j6INK4a
status of 100 resected lung tumors
16@NK4a—TotalPRB+/Pl&NK4af@@/@16INK4a±
pRB+/p16@@pRB_/pl6INK@Histology
Status16tNK4a±
(%)Adenoca23
@
(49)5
(24)7
(17)0
(0)47(100)Squamous10
(0)42(100)Adenosquamous1
(17)6(100)Large
cell(20)2
(100)Total3514
(11)
(17)
(0)
19 (40)0
25 (60)0
4 (67)1
(40)
(40)0
(0)j
501100pRB
+/p16@@pRBTotalStage
(%)I24
(2)@9(100)II8
(0)24(100)Ill2(13)2(13)
pRB
+/pl6INK4@pRB
(41)1
(33)1
1 (19)
(4)
23 (39)1
15 (63)0
12(75)0(0)16(100)IV(100)(0)
(100)Total3514
(0)0
(0)1
501100Correlation
of pRB and @16INK4a
status
pRBpl6INK4@35
pRB+
14pl6@4a_50
1P=O.00019
3416
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@
@
@
@
@
@‘@tt •@‘@:@2
• @q .
@.
S
@making
•‘
\
-@
S
,
—.
“@
•
. @â€S̃ I
‘@
Rb AND @16INK4a
EXPRESSIONIN NON-SMALLCELL LUNG TUMORS
absent*expression
study. Sixty-four percent of the tumors (P = 0.00019) showed
showingaberrant
of pRB or p16INK4a, with a single tumor (1%)
of0squamous
expression of both gene products (Table 2). The majority
adenosquamouscarcinomas
cell carcinomas (60%), as well as the few
A.
I.
%
.4.
*@
p
@
@
.4
;4
@
P
@
@‘
&
@
@
no detectable
p16INK4a protein
‘a
c?@iv
@
‘
?---@.@‘
ny―
@
..
@
:
:@1:
@.
@
: ,
:@.‘,:
.
‘
@•‘@‘ .
;:..:?.1
‘
•;, . .
‘@
‘@ @.‘
I
S.
,.@;:
@
@l'lv.
@‘ *44@
j
ijr
@
S ‘
@
,@
.
@...
‘‘@
Fui. I . In@cr'.e r@11nh111()@taIr1It1t.
ol pRB uul p I(@‘“
‘@ii NS('L( .:\r e@ectedadeii
@
carciIloIna
@
‘.taiiiiiig@trutiiaIetcinent@ arc @eciiiii the p 1(@ ‘@@tain@
( i,/@@t.
@
cd I@ are
@
expressed
.::
@
@
(83%),
In contrast, adenocarcinomas, which made up the largest histolog
subtype in this survey (47%), lacked p16INK4a protein expression
‘
,ical
4
:@.
i@―i@
recently,reported
less
often (40%). Similar results for adenocarcinomas were
.,
I
on a smaller cohort of patients with resected NSCLC (13). In
the present study, pRB expression was identified in all tumor
types.Fifteen
percent of the tumors demonstrated aberrant expression of the
Rb gene product. Although it was slightly more common for adeno
to possess a wild-type pRB staining pattern (89% posi
tive)
than
for
squamous cell carcinomas (83%), the difference was
1*
. ,.
@;@4carcinomas
small. The inverse correlation of pRB and pl6INK4a expression ob
4
..served
was more striking in the subset of squamous cell carcinomas.
s..—-.
,..@
0 • —@
Seventy six percent of the squamous cell cancers had lacked either
,
- . ,
#@ ,@ -@
pl65――@4―
or RB protein, whereas only 51 % of adenocarcinomas
displayed this molecular phenotype. If the adenocarcinomas are re
fl
a
_f@d@J@@
0
moved from analysis, 77% of the tumors from patients resected for
cure demonstrate absence of either wild-type pRB or p16IN@@4@@
protein
‘
@, ,1lb
*,
(Pinverse.
= 0.00029). There was a general increase in incidence of
@
@
@
(Table.2).
)%.I. :@.;
‘
from
a sta@!e I NS('I
C
_@or111)JctcI\ I1Ci.!Jtl\C
iiiiiuirio@taineJ
or pRB
( @1
) and
1(@‘
( B . I@@Iti\c'
‘@
icrca@the let )pIa@tIC
:
4( )(
@
,
@is‘•a@
.S:‘
: @..- : . . .
. ..
.@ .5 .. S
“@ S
:
‘
@
2
@
11
@
@
@
@1h*k
@
@P
,@,
@
..
@
.
@
@
@
t
, rS U
:
X200.:@•
@
@
-.5
.
,
.
*@-
.
a
@.
.
/,
‘@)
S
.
Fig. 3. Negative pl6@@@@a
immunostaining
@:.
v@ ‘
:@
.
.
:
:@
4r%@:@
5'
..,,
.
S
,t-
‘ .
@:
@@‘@_‘_S' .
i@
#
ø@ wR#@
I
@
111.11 II
2 I ()I_,III\ I1(15@
l I (IS
@S
I
p@itI\c \‘@(
I .( tHur
,I(IJ.I@_lilt
) .1 J@I (@
II @. i I I
It
ill'
w
l@
‘@@fL@
.
\ Ic@IIIcd @‘.‘(
I ( JCI1II1II\It.IIL\.11
I —11eit( I\
,
:
:‘
@
of NSCLC: a negative pl6INK4a NSCLC
:@
@
@
. ‘S
S, @•
.r
‘@
,@
.
@.‘ with pl6IN@@@positive
tumor
bronchiole (arrow).
S
e
$t@―b
.
,
:@:‘.;
S
11
•
portion of the tumor. X400.
@
.t
@
Ii,.,;
.,
@S\
:
@.
of pRB-negative disease was not seen in this study, although the
overall number of tumors with aberrant pRB expression was small,$
analysis of subsets difficult.
4. p16JN@@negativemetastatic lesion from a tumor with p16@4a@positive
elements. A patient with a primary tumor that was focally positive for pl6@@4a(Fig. 3)
The inverse correlation of RB protein and pl6INK4a expression
.
.
.
had a recurrence in the brain. The metastatic lesion was resected. Immunostaining for
previously reported in NSCLC cell lines (6) and tumors (1 1—13)was
pl6INK4a demonstrates the absence ofp16@4a protein. Positively staining glial elements
demonstrated in large series of resected early stage tumors in thisFig. are seen in the surrounding nonmalignant tissue (arrow). X400.
3417
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
Rb AND @161NK4a
EXPRESSIONIN NON-SMALL CELL LUNG TUMORS
B.
A.
I 00
I 00
c@
>
2:
Co
>
80
.@ 80
C,)
U)
C
C
a) 60
a)
a)
>
a)
2
60
a)
0.
0.
40
a)
>
@40
E
E
20
0
0
20
0
Fig. 5. Survival of patients undergoing thoracot
0
omy at MVAMC. A, survival of 100 patients Un
500
(n
1000
1500
2000
0
24), and ifi (n = 16) NSCLC is indicated by
stage. B, the same cohort of patients were examined
histology (C) and pRB status (present or absent; D),
Cl)
adeno
C
a)
e
60
60
a)
0.
0.
a)
a)
>
>
Co
40
Co
E
E
0
0
20
0
500
1000
1500
2000
40
20
0
expression of pRB and pl6iNK4a with stage. In stage I disease, 58% of
patients have inverse expression of pRB and pl6@4― protein, com
pared with 67% of stage II tumors and 88% of stage ifi disease (Table
2). The small number of tumors in each category makes the signifi
cance of this trend unclear, but in general, there is an increased
incidence of both absence of pl6'@4@' protein, as well as inverse
expression of pRB and pl6@@4a protein, with increasing stage of
disease.
The overall survival for this cohort of patients from the MVAMC
stage of the tumor is slighfly
worse than
that reported in the United States as a whole (Fig. 5A; Ref. 5). When
the patient survival in relation to pl6'@4@' status was analyzed, a
statistically significant difference in survival was seen for ail patients
(Fig. SB). Patients who are pl65@4@'negative had a worse prognosis
than those with pl6Th@4@@@positive
tumors (P = .0404). When further
analyzed by stage or histology, there was a consistent trend toward
in tumors
demonstrating
p16@'@4―positivity,
but it did
not reach a statistical significance (P < 0.05) in any of these cases
(data not shown). A possible explanation for the decreased survival
between
500
1000
Days
Days
better survival
2000
2: 80
C
e
a)
1500
Co
>
80
U)
a)
2000
100
Co
>
2:
1500
D.
I 00
with no significant survival difference noted.
to the pathological
1000
Days
C.
for survival by pl6@@(4astatus (present or absent;
P = .04). Survival analyses were also performed by
as related
500
Days
dergoing thoracotomy for stages I (n = 59), II
the p16@4L@@positive and -negative
subgroups
as an aggre
gate could be found in the high number of pl6@@4L@
tumors among the
squamous cell carcinoma histological subtype and the relatively lower
number
among
the adenocarcinomas.
However,
no significant
differ
ences in survival existed between these two histological subtypes in
this study (Fig. SC'). Cox regression analysis demonstrates that
pl6―@@status of the tumor was the only significant variable with
regard to overall survival identified in this study (P
.0317). Previ
ous studies have reported that pRB-negative tumors have a worse
overall prognosis, particularly among stage I and II patients (17). This
was not demonstrated in our analysis for either stage or for the group
as an aggregate (Fig. SD).
Discussion
This study provides strong in vivo confirmation of previously
published lung cancer cell line and tumor findings detailing the
inverse relationship between Rb and pJ6INK4a gene product expres
sion in NSCLC (6, 11—13).However, our finding of 65% of early
3418
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Rb AND @16LNK4a
EXPRESSIONIN NON-SMALL CELL LUNG TUMORS
stage tumors expressing either pRB or pl6@4a is slightly lower than
the rate of inverse correlation observed in lung cancer cell lines (6). In
a survey of NSCLC cell lines, 85% of the NSCLC cell lines demon
strated inverse expression of wild-type Rb andp16@―4―
gene products
when analyzed by immunoblot. Furthermore, a recent series of 61
NSCLC lung cancers of all stages also reported on the correlation of
pRB and p16K4L@ protein
status.
In that study,
84% of tumors
were
found to have inverse expression 0f@16INK4aand pRB (13). Although
immunohistochemistry can usually detect mutant RB protein based on
an abnormal staining pattern, it is possible that some of the pRB
“positive―
tumors we have identified will possess missense point
mutations or small deletions, resulting in a nonfunctional Rb gene
product that nonetheless retains normal staining (14). In general,
however, the frequency of these smaller mutations in Rb is relatively
low in NSCLC (4% in the largest series of cell lines examined; Ref.
18). The incidence of inactivating mutations in pl6LNK4ain NSCLC is
less well established. A recent report observed that 7% of NSCLC
tumors possessed p16@4a point mutations but that these mutations
were limited to patients with stage ifi and IV disease and not observed
in patients with stage I and H disease (19). Although our analysis may
underestimate the incidence ofpl6@4― and pRB inactivation, current
data suggest that there is a relatively small percentage of these tumors
that possess missense mutations of either the Rb or pJ6@―@4―
genes
that result in a gene product that continues to produce positive nuclear
staining in immunohistochemical assays.
Previous studies have reported that aberrant Rb gene product ex
pression is associated with a worse prognosis in NSCLC, particularly
stage I and H disease (17). Our study failed to demonstrate a similar
trend, although the number of pRB-negative tumors in the present
study (15%) was smaller than in the previously reported study (24%)
@
and thus limits the power to detect significant survival trends, partic
ularly among subgroups. In contrast, we found a strong association
between aberrant pl6@@4a expression and worse survival, which was
identified in all patients (Fig. SB). Given the inverse correlation
between Rb and pJ6@―4―
gene products, it would be interesting to
speculate whether the worse survival associated with lack of p16INK4a
expression reflects an unexpected positive survival advantage among
patients with pRB-positive tumors in this cohort of patients. No such
correlation was seen. The observation that lack 0f@16@4a expression
is associated with a worse prognosis is consistent with the increased
incidence of pl6@4a mutations observed in metastatic lesions.
p16@4a negativity has been correlated with advanced stage (stage Il
and IV) in previous studies, and
positive (wild-type) tumors
have been demonstrated to be associated with subsequent pl6@4I@
aberrant metastatic disease (19). The increased rate of loss of pl6'@41'
expression with higher-stage disease demonstrated in the current
study is consistent with these observations. The higher rate of loss of
pl6@@4a expression in advanced disease was also seen in a small
subset of tumors with focal pl6@4a positivity that had subsequent
pl6@4a@negative metastases by immunohistochemistry. The biolog
ical impact of loss of pl6INK4a in the development of metastatic
disease is not clear, but a similar loss of pRB expression has been seen
in advanced-stage NSCLC, giving further evidence that disruption of
the molecular block to S-phase is critical in the molecular pathogen
esis of this disease (20). Correspondingly, we identified an increase in
the inverse expression of pRB and @16@4awith advancing disease.
Although the absence of wild-type p16@4― expression has been
associated with advanced disease, loss of heterozygosity at the
pl6@4a locus (9p2l) remains a common finding in all NSCLC and
is associated with even the earliest stages ofcancer and dysplasia (21).
It is not surprising, therefore, that loss of expression of @16@4amay
be one of the most frequently acquired genetic mutations in this
disease. The observation that half of all early stage NSCLCs have no
detectable expression of pl6@―4@protein is consistent with the hy
pothesis that cdk4-mediated phosphorylation in a cell cycle-dependent
manner of Rb and Rb-related proteins is critical in the molecular
oncogenesis of NSCLC (6). Although a relatively large (35%) subset
of tumors in this study possess no detectable abnormalities in either
pl65@4―or pRB, and only 51% percent of the samples clearly lack
p16INK4@L
expression, it is clear that defects in the pathway responsible
for cdk-mediated phosphorylation of pRB are critical events that
occur in the majority of NSCLCs. Recent reports of both mutations in
cdk4,
resulting
in abrogation
of the pl6@4a@mediated
cdk4
inhibi
tion (22), as well as cycin D family (23) amplification in NSCLC,
strongly imply that aberrations in the other participating proteins in
this pathway are involved in the molecular carcinogenesis of lung
cancer. Moreover, additional mutations in a number of other molec
ular targets are commonly found in NSCLC, which also play a
significant role in the development and maintenance of the trans
formed state (2).
This study confirms the previously hypothesized importance of muta
tions of the pRB-cyclin-cdk4 pathway in NSCLC tumors. The observa
tion that lack ofpl6'@―@expression is associated with a worse prognosis
in a disease with a generally poor outcome and minimally effective
therapies is of uncertain clinical importance. With the advent of striking
survival advantages for neoadjuvant chemotherapy for locally advanced
stages of NSCLC
(stage ifiA; Refs. 24 and 25), it is possible that subsets
of patients with a worse prognosis, which may benefit from more ag
gressive combination therapy approaches, will be identified in the future
by molecular techniques (26). However, given the wide spectrum of
genetic targets already identified in NSCLC, it remains likely that no
single prognostic factor, gene target, or biochemical pathway will provide
a therapeutic or prognostic answer to the problem of NSCLC. Nonethe
less, aberrations of p16@N@@
and the associated S-phase: cdk pathway
likely represent one of the most common molecular defects in this
distressingly common form of cancer.
Acknowledgments
We thank David Allen, Sandra Frizelle, Patti Baribeau, and Vicki Morrison
for their assistance in obtaining and analyzing patient data.
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Rb and p16INK4a Expression in Resected Non-Small Cell Lung
Tumors
Robert A. Kratzke, Todd M. Greatens, Jeffrey B. Rubins, et al.
Cancer Res 1996;56:3415-3420.
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