Blood Group Antigen A and Flow Cytometric Analysis in...

Vol. 3, 87-93, January 1997 Clinical Cancer Research 87

3 The abbreviation used is: NSCLC, non-small cell lung cancer.

Blood Group Antigen A and Flow Cytometric Analysis in Resected

Early-Stage Non-Small Cell Lung Cancer’

Stephen L. Graziano,2 Arthur H. Tatum,

Nick J. Gonchoroff, Nancy B. Newman,

and Leslie J. KohmanDepartments of Medicine [S. L. G., N. B. N.], Pathology [A. H. 1.,N. J. G.l, and Surgery [L. J. K.], Veterans Affairs Medical Center andState University of New York Health Science Center, Syracuse,New York 13210

ABSTRACTThe loss of blood group antigen A on tumor tissue has

been reported to be a strong adverse prognostic marker for

patients with resected non-small cell lung cancer (NSCLC).Results have varied with respect to the prognostic signifi-cance of flow cytometric data. We sought to confirm the

prognostic significance of blood group antigen A loss and

flow cytometry in a large cohort of patients with early-stageNSCLC. Two hundred and sixty patients with surgically

resected stage I (n 193) and II (n = 67) NSCLC with at

least a 5-year follow-up were identified. Using paraffin-

embedded primary tumor, immunohistochemical stains for

blood group antigen A were performed on 90 patients withblood type A or AB. The DNA index and percentage of cellsin S phase were successfully obtained on 188 and 152 pa-tients, respectively.

The median survival time of the patients with primary

tumors negative for blood group antigen A was 38 months(n = 36), compared with 98 months (ii 54) for those with

antigen A-positive tumors (P < 0.01). The median disease-free survival times for antigen A-negative and -positive tu-

mors were 26 months and 98 months, respectively (P <

0.01). The median survival time of the patients with aneu-

ploid tumors was 51 months (n = 131), compared with 50months (n = 57) for those with diploid tumors (P 0.42).The median survival time of the patients with S phase >8%was 44 months (n 105), compared with 60 months (n 47)

for those with S phase �8% (P = 0.18). Multivariate anal-

ysis showed that the loss of antigen A, higher N and T stages,

and the presence of mucin predicted for poorer disease-freeand overall survival.

In the subgroup of patients with blood group A or AB,the loss of A antigen was the most powerful negative pre-

Received 2/26/96; revised 10/7/96; accepted 10/I 7/96.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I Supported by the Veterans Affairs Research Service.2 To whom requests for reprints should be addressed, at Veterans AffairsMedical Center, 800 Irving Avenue, Syracuse, NY 13210. Phone: (315)

476-7461; Fax: (315) 464-8206.

dictor of survival. Aneuploidy and percentage of cells in S

phase were not of prognostic significance in this group of

patients with resected stage I and II NSCLC. The value of

blood group antigen A analysis needs to be evaluated inlarger and prospective studies of early-stage NSCLC. Alter-ation of blood group antigen cell surface expression may

represent an important marker for more aggressive biolog-ical and metastatic behavior in NSCLC.

INTRODUCTIONLung carcinoma is the leading cause of cancer-related

deaths among both men and women in the United States. In

1996, it is estimated that 159,000 deaths will occur from lung

cancer (I). Clinically and therapeutically, lung cancer is gener-

ally divided into small cell lung cancer and NSCLC.3 For small

cell lung cancer, which comprises 20-25% of all cases, com-

bination chemotherapy forms the cornerstone of therapy. For

NSCLC (e.g., squamous cell carcinoma, adenocarcinoma, and

large cell carcinoma), surgery is the major curative modality for

patients without demonstrable metastatic disease (2).

The well-established negative prognostic factors for

NSCLC include higher stage, weight loss, poor performance

status, and presence of systemic symptoms (2). Other studies

have reported the possible prognostic impact of tumor size (T�

versus T,; Refs. 3 and 4), sex, age, histological subtype (squa-

mous versus non-squamous; Ref. 3), degree of differentiation

(5), vascular invasion (5), mucin expression (6, 7), aneuploidy

(8, 9), K-ras mutation and expression (10, 1 1), p53 mutations

(12), increased pl8S�’ expression (13), and bcl-2 expression

(14).

The expression of ABH blood group antigens is found in a

variety of epithelial cells, including urothelium, gastrointestinal,

mucosa, and lung, as well as RBCs. An early study by Kay and

Wallace suggested that the loss of A and B antigens on urothe-

hal tumors was correlated with more aggressive behavior (IS).

Supporting evidence was provided by Limas et a!., showing a

favorable prognosis for transitional cell carcinomas of the uri-

nary bladder with retention of blood group antigens A, B, and H

(16).

Because the expression of blood group antigens in tissue

depends on blood type, Lee et al. studied the survival of patients

with NSCLC within the context of their ABO blood type (17).

Of 164 patients who underwent curative surgery, 71 were blood

type A or AB. Survival of the 28 patients who had primary

tumors negative for blood group antigen A was significantly

shorter than that of the 43 patients with positive tumors (P <

0.001) or the 93 patients with blood type B or 0 (P 0.002).

Another study correlated clinical course of patients with

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88 Prognostic Importance of Antigen A and Flow Cytometry

NSCLC with immunohistochemical staining of their tumors

with a monoclona! migration-inhibiting antibody (MIA-1S-S)

directed against H/LeY/Leb antigens (18). Enhanced expression

of these antigens in tumors is associated with the absence of A

and B blood group antigens. Among 149 patients studied, S-year

survival in the 91 patients whose tumors stained with MIA-1S-S

was significantly worse than survival in the 58 patients whose

tumors showed no MIA-lS-S staining (21 versus 59%; P <

0.001). These studies suggest that tumor-associated carbohy-

drate antigens may be an important marker for invasiveness and

metastatic potential in patients with NSCLC (19).

A number of studies have reported that DNA content

(aneuploid versus diploid) is prognostic for various groups of

patients with NSCLC (8, 9, 20-22). Others have reported that a

high percentage of cells in S phase is associated with poorer

survival (22-25). However, the interpretation of many of these

studies is limited by small numbers, heterogeneous groups, and

subset analysis. In addition, many studies have failed to detect

prognostic significance for flow cytometric data (26-29). Thus,

although flow cytometric data have been one of the most com-

monly studied of all prognostic variables in early-stage NSCLC,

definite conclusions regarding their prognostic importance can-

not be drawn.

The purpose of the present study was to investigate the

prognostic significance of flow cytometric data (DNA index and

S phase) and loss of blood group antigen A in a large cohort of

well-characterized patients with resected stage I and II NSCLC.

MATERIALS AND METHODS

Two-hundred and sixty patients with stage I and II NSCLC

who underwent curative-intent surgery at least S years earlier,

with available follow-up information, were identified for study

from four hospitals in Syracuse, New York. These included

University Hospital (State University of New York Health Sci-

ence Center at Syracuse), Veterans Affairs Hospital, Crouse-

Irving Memorial Hospital, and St. Joseph’s Hospital. Local

Institutional Review Board approval was obtained for these

studies. The patients were identified from the tumor registries,

operating room records, and records of individual thoracic sur-

geons. Cases were excluded if there were two primary tumors at

the time of diagnosis or if the patient did not leave the hospital

after surgery, died within 30 days of surgery, or did not meet the

definition of stage I (T, or T2, N0M0) or II (T1 or T,, N,M0)

according to the International Staging System (30). Once iden-

tified, the patient charts, including pathology, operative reports,

and blood type were reviewed, and the data were coded as

described previously (7). The characteristics of the 260 cases are

shown in Table 1.

The pathology report and one representative formalin-

fixed, paraffin-embedded block of the primary tumor were ob-

tamed for each case. Four-�.am slides were sectioned from each

patient block and placed on Probe-On Plus slides (Fisher Sci-

entific, Rochester, NY) to facilitate performing the immunohisto-

chemical studies on an automated stainer (Code-On Automated

Immunohistochemical Analyzer DNA Unit; Instrumentation Lab-

oratories, Lexington, MA). In addition, three S0-�im sections were

cut for flow cytometry studies. Sections were stained with H&E to

confirm the original pathological diagnosis. In addition, a com-

Table 1 Patient characteristics (n = 260)

AgeMedian

Range63.5

(40-94)

Sex

FemalesMales

Stage

I

T,NQMOT,N#{216}MO

IIT,N,M#{216}

T2N,M0Surgery

LobectomyBilobectomyPneumonectomySegmentectomyWedge resection

Weight loss<5�5 lbs

UnknownMediastinoscopy

YesNo

Unknown

76 (29%)184 (71%)

193 (74.2)

81

112

67 (25.8)7

60

18 1 (69.6)13 (5.0)39 (15.0)13 (5.0)14 (5.4)

199 (76.5)

38(14.6)

23 (8.8)

173 (66.5)86 (33.1)

1 (0.4)

bined staining method for keratin and mucin-like substances

adapted from Kreyberg was performed in all cases (31). This

system uses alcian blue to stain mucin and erythrosin B to stain

keratin (32).

All studies were performed without knowledge of patient

outcome. A modification of the avidin-biotin complex technique

was used for immunohistochemica! studies (33). This involved

employing a labeled streptavidin-biotin technique utilizing a

hybrid system based upon commercially available reagents. This

system was developed in the Immunopathology Laboratory at

the State University of New York Health Science Center

(A. H. T.). Slides were deparaffinized and rehydrated through

xylene to graded alcohol to buffer solution.

Blood Group Antigens. Studies for antigen A were per-

formed on all patients with blood type A or AB. The blood type

was unknown for 1 1 patients, and tumor tissue from these

patients was stained for antigens A and B to determine their

blood type (see below).

The slides were incubated with normal goat serum (I %

normal serum in hybrid diluent) for S mm at 42#{176}C.Hybrid

diluent was 2% FCS in 1 X Automation Buffer [a I : 10 dilution

of lOX Automation Buffer, a commercially available Tris buffer

from Biomeda Corp. (Foster City, CA)]. By capillary gap ac-

tion, the excess blocking solution was removed, and the mono-

clonal primary antibody, blood group A antibody (Biogenex

Corporation, San Ramon, CA) at 1 :50 dilution in Primary An-

tibody Buffer (proprietary reagent available through Biomeda

Corp.), was incubated at room temperature for 30 mm.

The slides were then washed twice in I X Automation

Buffer, incubated sequentially with a 1:200 dilution of goat

biotinylated antimouse 1gM (Vector Laboratories, Burlingame,

CA) in hybrid diluent for 10 mm at 42#{176}C,washed in I X



Clinical Cancer Research 89

Automation Buffer, and then incubated with a 1 :80 dilution of

alkaline phosphatase-conjugated streptavidin (Biogenex Labo-

ratories) in hybrid diluent for S mm at 42#{176}C.The slides were

then washed twice in 1 X Automation Buffer and stained as

specified by the Fast Red Substrate Pack (Biogenex Laborato-

ries), washed twice in a solution of 25% absolute methanol in

distilled water, and counterstained with hematoxylin. The slides

were then washed sequentially with 1 X Automation Buffer and

25% absolute methanol in distilled water and coverslipped with

a synthetic medium. A negative control slide was included in

which I X Automation Buffer was substituted for the primary

antibody. Endothelium and RBCs were used as internal positive

controls. The same procedure was performed on tissue from the

1 1 patients with unknown blood type using a monoclonal anti-

body for the blood group B antigen (Biogenex Corp.) at a I :40

dilution.

Sections were evaluated by a pathologist (A. H. T.) without

knowledge of patient outcome. The results were graded from 0

to 4 according to intensity of staining and from 0 to 4 by

percentage of cells positive as follows: 0, 0%; 1, <25%; 2,

25-<S0%; 3, S0-<75%; and 4, 75-100%. Positives were con-

sidered �2 staining in any percentage of cells.

Flow Cytometry. Each paraffin-embedded sample was

dewaxed by the method described by Hedley et a!. (34), and the

extracted cell nuclei were stained using propidium iodide fol-

lowing a method developed by Vindelov (35). The DNA index

and percentage of cells in S phase were determined as described

previously (36). For each daily batch of 18 specimens, controls

included a tissue sample that gave a DNA diploid histogram and

a sample that gave a DNA aneuploid histogram. Both normal

and abnormal controls were monitored daily, and the instru-

ments were aligned daily and checked with a calibrator to detect

instrument drift. Tissue sections were cut from before and after

the three 50-�i.m sections to ensure that a high percentage

(>20%) of the material tested was tumor.

Statistical Considerations. The major end point of this

study was to assess whether the presence of each of the

markers would predict for survival and disease-free survival.

Survival time was defined as the time from the date of

surgery until death from any cause. Disease-free survival was

defined as the time from surgery until the time of tumor

recurrence or date of last follow-up. An observation was

censored at the date of death due to other causes or date of

last follow-up. Life probability calculations were performed

using the Kaplan-Meier method (37) and the Wilcoxon Test

to detect differences in survival curves (38). The clinical,

pathological, and biological variables were tested for associ-

ation with survival and disease-free survival (39). Cox’s

proportional hazard models were employed to determine the

impact of patient characteristics on survival and disease-free

survival (40). For purposes of analysis, categorical variables

were broken into dichotomous groups. Forward stepwise Cox

regression was performed to calculate the joint effect of

covariates on survival and disease-free survival. Covariates

entered the model if they were considered an important

variable a priori or met a level of significance of 0.25 and

remained in the model at a significance of 0.15.

RESULTSThe characteristics of the 260 patients are shown in Table

I . The median age was 64 years, and 7 1 % of patients were

males. One hundred and ninety-three (74.2%) patients were at

stage I, and 67 (25.8%) were at stage II. Lobectomy was

performed in 69.6% of cases, and pneumonectomy in 15.0%.

Mucin was present in 79 cases (30.4%), and keratin was present

in 61 cases (23.5%). The degree ofdifferentiation was well in 30

(11.5%), moderate in 95 (36.5%), and poor in 135 (52.0%)

cases. The histological subtypes were as follows: adenocarci-

noma, 41.9%; squamous cell, 28.1%; large cell anaplastic,

16.2%; adenosquamous, 10.4%; and bronchioloalveolar, 1.9%.

The median survival times for the stage I (n = 193) and stage II

(n 67) patients were 67 and 33 months, respectively (Fig. IA;

P < 0.001). The 3- and S-year survival rates were 67 and 46%

for stage I and 49 and 28% for stage II patients, respectively

(Fig. 1A).

Tumor tissue from 1 1 cases with unknown blood type were

stained with blood group antibodies A and B. Six were negative

for RBC staining with both antibodies, and these were assumed

to be blood type 0. Of the five remaining cases, four were

identified by red cell staining for antigen A, and one was

positive for antigen B. Thus, with the additional in vitro infor-

mation, the blood types of the 260 patients were as follows: 94

were blood type A, 8 were blood type AB, 30 were blood type

B, 125 were blood type 0, and 3 had unknown blood types. Of

the 102 patients with blood type A or AB, 12 could not be

analyzed, because no tumor remained in the block (7), or tissue

was poorly fixed (5). This left 90 cases that could be analyzed

for loss of blood group antigen A.

Of the 90 patients who were analyzed, 54 (60%) demon-

strated positive tumor staining for blood group antigen A. For

patients with stage I NSCLC, 41 of 70 (59%) tumors were

positive, and for stage II NSCLC patients, 1 3 of 20 (65%) were

positive. Disease-free and overall survival were strongly asso-

ciated with the presence of blood group antigen A on tumor

tissue for patients with stage I NSCLC (Table 2; Fig. 1 , B and

C). For stage I patients, the median survival time was 98 months

for antigen A-positive cases (n 41) and 41 months for antigen

A-negative cases (n = 29). The S-year survival rates were 61

and 3 1 % for antigen A-positive and -negative cases, respec-

lively (Fig. lB).

There was no significant difference in overall or disease-

free survival for patients included in the blood group antigen A

study (n = 90) versus those excluded (n = 170). The median

survival time for patients with blood type B or 0 was 78.4

months compared with the blood type A or AB patients with

antigen A-positive tumors (98 months; P = 0. 13) or antigen

A-negative tumors (38.4 months; P 0.03). The mean tumor

size for antigen A-positive tumors was 3.78 cm compared to

3.70 cm for antigen A-negative tumors (P 0.84). In addition,

there was no apparent influence of histology on antigen A status.

Twenty-seven of 44 (61%) adenocarcinomas were antigen A

positive, and 17 of 30 (57%) squamous cell carcinomas were

antigen A positive. For squamous cell carcinoma, the median

survival time for patients with antigen A-positive tumors was

>79 months (n = 17) versus 27 months (n = 13) for those with

antigen A-negative tumors (P < 0.01). For adenocarcinoma, the



MONTHS SURVIVAL

MONTHS SURVIVAL

MONTHS SURVIVAL


A

B

z>>

U)z0I-0

Li�

C

0z>>

DU)z0

0z>>

(I)z0I-

C)

U.

Fig. 1 A, overall survival curves for patients with Stage I and IINSCLC (P < 0.001). B, overall survival curves for patients with StageI NSCLC for antigen A-positive and -negative tumors (n = 70; P <

0.0!). C, disease-free survival curves for patients with Stage I NSCLCfor antigen A-positive and -negative tumors (P < 0.01).

Table 2 Overall and disease-free survival by blood group antigen A

data

A’� A P value

Median overall survival in months (N)Stage I 98 (41) 41 (29) <0.01Stagell 70(13) 37(7) 0.32All patients 98 (54) 38 (36) <0.01

Median disease-free survival in months (N)Stage I 98 (41) 26 (29) <0.01

Stagell 37(13) 36(7) 0.66

All patients 98 (54) 26 (36) <0.01

Table 3 Overall and disease-free survival by flow cytometry data

Median overall survival in months (N) P value

Diploid AneuploidStage I 98 (44) 98 ( 101 )Stage II 14(13) 34(30)All patients 50(57) 51 (131)

S phase �8% S phase >8%

Stagel 99(39) 100(77)

Stage II 14 (8) 26 (28)All patients 60 (47) 44 (105)

0.420.260.42

0.140.240.18

Median disease-free survival in months (N) P value

Diploid Aneuploid

Stage I 98 (44) 99 (101)Stage II 11 (13) 24(30)All patients 41 (57) 34 (131)

S phase �8% S phase >8%

Stagel 100(39) 66(77)Stage II 10 (8) 14 (28)All patients 47 (47) 26 ( 105)

0.410.140.87

0.12

0.360.13

median survival time for patients with antigen A-positive tu-

mors was 70 months (n = 27) versus 41 months (n = 17) for

those with antigen A-negative tumors (P = 0.10).

As expected, the recurrence rate was higher in the antigen

A-negative group (75%) compared with the antigen A-positive

group (42%; P = 0.005). There was no difference in the pattern

of first recurrence (distant versus local) or the cause of death. In

the antigen A-positive group, 59% of the recurrences were

distant and 41% were local compared with 64 and 36%, respec-

tively, in the antigen A-negative group (P = 0.73).

For DNA ploidy, 72 cases could not be analyzed because of

the use of the mercury-based Bouin’s fixative (28 cases); mad-

equate amount of tumor available (25 cases); or technical prob-

lems, such as poor formalin fixation, less than 20% of tissue

tumor, or poor histogram (19 cases). Thus, of the total number

of patients, 1 88 specimens were analyzed successfully for DNA

ploidy. For the percentage of cells in S phase, 108 cases could

not be analyzed because of the use of the mercury-based

Bouin’s fixative (28 cases); inadequate amount of tumor avail-

able (25 cases); or technical problems such as poor histograms,

poor formalin fixation, or a third peak obstructing the S-phase

area (55 cases). Thus, 152 specimens were successfully ana-

lyzed for the percentage of cells in S phase.

We found no significant difference in median disease-free

or overall survival time for diploid versus aneuploid tumors or



1.0

AS�I��h�

I p..42 I

B

0z>>

C’)

z0

0

U.

1 ,O

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

��1. L

� S-PHASE

S-PHASE�0�”��-’

IP=.14 1

10 20 30 40 50 60

MONTHS SURVIVAL

Fig. 2 Overall survival curves for patients with Stage I NSCLC foraneuploid versus diploid tumors (P 0.42). B, overall survival curves

for patients with Stage I NSCLC for S phase �8% versus >8%

(P = 0.14).

U Survival analyses performed with N stage. T stage. age. mucin,

and the additional variable. There are three separate models, becauseindividual data were not available on all cases.

b RR, relative risk.


A

0.9

0.8�

0z o.7�>� 0.6’

� 0.5’

0I- 0.4

� 0.3

0.2

0.1�

0.0W

0 10 20 30 40 50 60

MONTHS SURVIVAL

for S phase �8% versus >8% (Table 3; Fig. 2). When squa-

mous histology alone was analyzed, there was no significant

difference in survival (P = 0.67) or disease-free survival (P =

0.32). Even the subgroup of diploid tumors with a low percent-

age of cells in S phase showed no survival differences (P =

0.49).

Survival analyses were performed to test for differences in

overall and disease-free survival between patients having a

positive or negative marker. A!! variables found to be significant

in univariate analysis (P < 0. 10) were included in the multiva-

riate analysis. The following covariates were included in the

analyses to assess their effect on survival and disease-free

survival: antigen A (presence versus absence); DNA index

(diploid versus aneuploid); percentage of cells in S phase (�8%

versus >8%); N stage (N0 versus N,); T stage (T, versus

age; and mucin (Table 4). Both univariate and multivariate

Table 4 Univariate analyse(

s for overall and

P values)

disease-free survival

Overall survival Disease-free survival

N stage (N, vs. N0)T stage (T2 vs. T,)

Antigen A (- vs. +)

Mucin (+ vs. -)

AgeS phase (>8% vs. �8%)

Ploidy (aneuploid vs. diploid)

<0.001<0.01

<0.010.02

0.100.18

0.42

0.02

<0.001<0.01<0.01

0.620.13

0.87

Table 5 Multivariate analyses f or overall and di sease-free survival”

Overallsurvival RR”

Disease-freesurvival RR

Antigen A (N = 90)Antigen A (- s’s. +)

T stage (T2 vs. T,)

N stage (N, vs. N,,)

Mucin (+ vs. -)

Age

S phase (N = 152)

N stage (N, vs. No)Mucin (+ vs. -)

AgeI stage (T, vs. T,)S phase (>8% vs. �8%)

Ploidy (N = 188)

N stage (N, vs. N,))Mucin (+ vs. -)

T stage (T2 vs. T,)

AgePloidy (aneuploid vs. diploid)

<0.01

0.02

0.06

0.08

0.83

<0.0010.02

0. 170.380.77

<0.0010.01

0.03

0.080.20

2.61

2.37

1.89

1.77

1.00

3.17

1.72

I .021.291.08

2.66

1.73

1.74

1.021.36

<0.01

0.03

0.10

0.01

0.73

<0.001<0.01

0.560.390.65

<0.0010.01

0.03

0.270.17

2.24

2.10

1.72

2.18

0.99

2.721.80

1.001.261.13

2.271.78

1.71

1.011.37

analysis revealed that the absence of antigen A, higher N and T

stages, and the presence of mucin predicted for poorer overall

and disease-free survival (Tables 4 and 5).

DISCUSSION

The present study was designed to explore the prognostic

impact of flow cytometry and blood group antigen A analysis in

patients with stage I and II NSCLC. The prognostic factors that

were important for survival in this group of patients by multi-

variate analysis include the loss of antigen A, higher N and T

stages, and the presence of mucin.

Aneuploidy and percentage of cells in S phase were not of

prognostic significance in this group of patients with resected

stage I and II NSCLC. There was clearly no impact of ploidy in

the present analysis. There was a trend toward poorer survival in

patients with cells in S phase >8%, but this did not reach

statistical significance. Our experience adds to the growing

literature of flow cytometric parameters in NSCLC.

In the present study, for patients with blood type A or AB,

the loss of blood group antigen A from tumor was a powerful

negative prognostic factor for disease-free and overall survival.

This study confirms the previous finding of Lee et a!. (17), but




in a larger group of stage I patients. Lee et al. studied 27 stage

I and 18 stage II patients, and the present study looked at 70

stage I and 20 stage II patients (17). However, recent studies by

Rice et a!. (41 ) and Dresler et a!. (42) failed to show prognostic

value for the loss of blood group antigen A in patients with stage

I NSCLC.

A number of studies show that blood group antigen A

expression may decrease in malignant cells (15, 43, 44). With

the decline in blood group antigen A, there appears to be a

corresponding increase in the levels of its biosynthetic precur-

sor, the H antigen (45). It is hypothesized that the absence of

antigen A is caused by the loss of activity of A transferase and

is followed by fucosylation of the immediate precursor of A

(namely H). Thus, the loss of antigen A is accompanied by an

increase in the expression of Lewis� (46). The Lewis3’ antigen

shares the same precursor in the biosynthetic carbohydrate path-

way, with the addition of a-1,3-fucose (44). The accumulation

of antigen H may contribute to the enhanced expression of

Lewis� observed in malignancies (45). In patients with NSCLC,

increased expression of Lewis�’ has been associated with a lower

survival rate (18).

Tumor-associated carbohydrate antigens (as defined by

blood group antigens) may have an important role in defining

invasiveness and potential for metastases of human cancers.

Normal biological processes that mimic activities in the meta-

static process in cancer also involve these carbohydrate anti-

gens. During wound healing, epithelial cells adjacent to the

wound exhibit loss of blood group antigens A and B. After the

healing process is over, the antigens are expressed again (47,

48). Also, during embryonic development of lung buds, various

Lewis antigens, some of which are also found in lung carci-

noma, are expressed at certain stages (49). Finally, two mono-

clonal antibodies developed by Miyake et a!. (18) showed

inhibitory action on the motility and metastatic potential of

malignant cells. These antibodies are directed toward the car-

bohydrate structure that is common to the H, Le�, and Le’�

antigens (18).

In summary, the present study confirms that the loss of

blood group antigen A is a powerful negative prognostic factor

for our group of surgically treated patients with stage I NSCLC.

Additional studies in similar groups of patients should be per-

formed to validate these conclusions. Although these findings

are important, this prognostic marker would be useful only in

the approximately 45% of the patients who have blood type A or

AB (SO). If alterations in the Lewis system antigens were found

to be prognostic, then this would be applicable to all patients

with NSCLC. We are presently investigating the prognostic

impact of the presence of the Lewis�’ antigen on tumor tissue in

this group of patients.

ACKNOWLEDGMENTSWe thank John Daucher and Gary Gamble for technical assistance.

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1997;3:87-93. Clin Cancer Res S L Graziano, A H Tatum, N J Gonchoroff, et al. early-stage non-small cell lung cancer.Blood group antigen A and flow cytometric analysis in resected

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