VOLUME

26

NUMBER

11

APRIL

10

2008

JOURNAL OF CLINICAL ONCOLOGY

O R I G I N A L

R E P O R T

Occult Axillary Node Metastases in Breast Cancer Are

Prognostically Significant: Results in 368 Node-Negative
Patients With 20-Year Follow-Up
Lee K. Tan, Dilip Giri, Amanda J. Hummer, Katherine S. Panageas, Edi Brogi, Larry Norton, Clifford Hudis,
Patrick I. Borgen, and Hiram S. Cody III
From the Department of Pathology;
Department of Epidemiology and
Biostatistics; Division of Breast Oncology, Department of Medicine; Breast
Service, Department of Surgery; and
the Memorial Sloan-Kettering Cancer
Center, New York, NY.
Submitted May 17, 2007; accepted
January 8, 2008; published online
ahead of print at www.jco.org on
March 10, 2008.
Supported by a grant from the Directors Fund, Evelyn H. Lauder Breast
Center.
Presented in part at the 38th Annual
Meeting of the American Society of
Clinical Oncology, May 18-21, 2002,
Orlando, FL; and Cody HS III, Borgen
PI, Tan LK: Ann Surg Oncol 11:227S230S, 2004.
Authors disclosures of potential conflicts of interest and author contributions are found at the end of this
article.
Corresponding author: Hiram S. Cody
III, MD, Breast Service, Department of
Surgery, Memorial Sloan-Kettering
Cancer Center, 1275 York Avenue,
New York, NY 10021; e-mail:
codyh@mskcc.org.
2008 by American Society of Clinical
Oncology

Purpose
In breast cancer, sentinel lymph node (SLN) biopsy allows the routine performance of serial
sections and/or immunohistochemical (IHC) staining to detect occult metastases missed by
conventional techniques. However, there is no consensus regarding the optimal method for
pathologic examination of SLN, or the prognostic significance of SLN micrometastases.
Patients and Methods
In 368 patients with axillary node-negative invasive breast cancer, treated between 1976 and 1978
by mastectomy, axillary dissection, and no systemic therapy, we reexamined the axillary tissue
blocks following our current pathologic protocol for SLN. Occult lymph node metastases were
categorized by pattern of staining (immunohistochemically positive or negative [IHC],
hematoxylin-eosin staining positive or negative [H&E]), number of positive nodes (0, 1, 1),
number of metastatic cells (0, 1 to 20, 21 to 100, 100), and largest cluster size ( 0.2 mm
[pN0i], 0.3 to 2.0 mm [pN1mi], 2.0 mm [pN1a]). We report 20-year results as overall survival
(OS), disease-free survival (DFS), and disease-specific death (DSD).
Results
A total of 23% of patients (83 of 368) were converted to node-positive. Of these, 73% were 0.2
mm in size (pN0i), 20% were 0.3 to 2.0 mm (pN1mi), and 6% were more than 2 mm (pN1a). On
univariate and multivariate analysis, pattern of staining, number of positive nodes, number of
metastatic cells, and cluster size were all significantly related to both DFS and DSD. On
multivariate analysis, each of these measures had significance comparable to, or greater than,
tumor size, grade or lymphovascular invasion.
Conclusion
In breast cancer patients staged node-negative by conventional single-section pathology, occult
axillary node metastases detected by our current pathologic protocol for SLN are prognostically significant.
J Clin Oncol 26:1803-1809. 2008 by American Society of Clinical Oncology

0732-183X/08/2611-1803/$20.00
DOI: 10.1200/JCO.2007.12.6425

INTRODUCTION

Sentinel lymph node (SLN) biopsy has largely replaced axillary lymph node dissection (ALND) as
the preferred method of lymph node staging for
breast cancer at many centers in the US and worldwide. Viewed as a surgical technique, an extensive
literature (including 69 observational studies1 and
four randomized trials2-5 of SLN biopsy validated by
a backup ALND) has established that SLN biopsy is
feasible, accurate, safe, less morbid than ALND, and
suitable for virtually all patients with stage I to IIIa
invasive disease. Viewed as a pathologic technique,
SLN biopsy allows the routine performance of
lymph node serial sections and/or immunohisto-

chemical stains, methods that have helped to

validate the SLN hypothesis,6,7 increase staging accuracy,8 reduce false-negative rates,9 and enhance
the prediction of non-SLN status in SLN-positive
patients.10 These same methods have also fostered
intense debate over (1) the proper method for
pathologic examination of SLN, and (2) the prognostic significance of SLN micrometastases, especially those detected only by IHC staining or as
isolated tumor cells.11 There is to date no consensus
on either issue, and here we address both. In a cohort
of 368 patients with node-negative invasive breast
cancer treated between 1976 and 1978 at our institution by mastectomy, ALND, and no systemic therapy, we have restaged the negative axillary nodes
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1803

Tan et al

using our current pathologic protocol for SLN biopsy, and we report
the results with 20-year follow-up.
PATIENTS AND METHODS
Patient Population
Over a 2-year period (1976-1978), 744 consecutive unselected nodenegative breast cancer patients were treated on the Breast Service, Department
of Surgery, at Memorial Sloan-Kettering Cancer Center (MSKCC; New York,
NY); 519 had invasive mammary adenocarcinoma and no prior history of
cancer, were treated by mastectomy (radical or modified radical) with an
axillary lymph node dissection (ALND) yielding at least five lymph nodes, and
received no systemic or radiation therapy. Paraffin blocks were available for
review in 368 of the 519 patients, and these 368 comprise our study cohort.
This study was conducted under a Waiver of Authorization from our institutional review board.
Pathologic Review
All diagnoses were initially rendered by MSKCC pathologists, and the
original tumor slides were available for rereview in 80% of cases (294 of 368),
confirming the diagnosis of invasive carcinoma, and noting tumor size, histologic type, multifocality, and the presence of lymphovascular invasion (LVI).
Invasive ductal carcinomas were graded by the modified Bloom-Richardson
system.12 The patient and tumor characteristics among patients with missing
axillary blocks (n 151) or missing primary tumor slides (n 74) were
substantially similar to those of the fully reviewed cases.
Reanalysis of Axillary Nodes
For all 368 patients, a total of 2,470 paraffin blocks from the axillary
lymph nodes were re-examined using our current pathologic protocol for
SLN, as described in detail previously.13,14 From each paraffin block, two
adjacent 5-m sections were cut at each of two levels 50 m apart. At each
level, one slide was stained with hematoxylin-eosin (H&E) and the other with
immunohistochemistry (IHC), using the anticytokeratin AE1:AE3 (Ventana
Medical Systems Inc, Tucson, AZ), for a total of four slides per block.
The H&E sections were reviewed and the findings recorded before review
of the IHC sections. In cases with isolated IHC-positive cells or microscopic
clusters, the cytologic features of the micrometastases (size and nuclear features) were compared with those of the primary tumor, and were accepted as
metastases only if they were concordant. Nodal metastases were evaluated for
(1) pattern of spread (single cells versus clusters); (2) size of the largest individual cluster to the nearest 0.1 mm; (3) location (subcapsular versus parenchymal), and (4) total number of tumor cells present. When metastases were
found in both levels or in multiple nodes, the number of tumor cells at each
level of all involved nodes was added up to estimate total tumor burden.
Each patient was categorized by pattern of staining (IHC/H&E,
IHC/H&E, and IHC/H&E), number of positive nodes (0, 1, 1),
estimated total number of metastatic cells (0, 1 to 20, 21 to 100, 100), and
size of largest metastatic cluster ( 0.2 mm [pN0i], 0.3-2.0 mm [pN1mi],
2 mm [pN1a]), following the most recent American Joint Committee on
Cancer (AJCC) Staging Manual (ed 6).15
Statistical Methods
The study end points were overall survival (OS), disease-free survival
(DFS) and disease-specific death (DSD). OS was calculated from date of
mastectomy to date of death or last-follow-up, and DFS was calculated from
the date of mastectomy to date of breast cancer recurrence or last follow-up.
DSD was calculated from date of mastectomy to date of death resulting from
breast cancer, and death resulting from other causes (approximately half of all
deaths) was treated as a competing risk. For OS and DFS, Kaplan-Meier
methods were used to estimate survival rates, and Cox proportional hazards
models were used to estimate relative risk and to fit multivariate models. For
DSD, the cumulative incidence for the competing risk model was estimated
using the methods of Gray,16 and competing risk regression was used to fit
multivariate models.17 All statistical analyses were performed using R (http://
www.r-project.org/) and SAS Software (SAS Institute, Cary, NC).
1804

RESULTS

Patient Population
The clinicopathologic features of the cohort are representative of
contemporaneous node-negative breast cancer patients (Table 1).
Seventy-five patients died as a result of other causes, 76 died as a result
of (or with) disease, and 217 were disease free at last follow-up. Median

Table 1. Patient Characteristics

Variable
Age, years
Median
Range
50
50
Laterality
Right
Left
Tumor type
Ductal
Lobular
Grade
I
II
III
Lobular
Missing
Multifocality
Absent
Present
Lymphovascular invasion
Absent
Present
Tumor size, cm
1.0
1.1-2.0
2.0
Missing
Converted to node
positive
No
Yes
Pattern of staining
IHC/H&E
IHC/H&E
IHC/H&E
No. of nodes positive
0
1
1
No. of metastatic cells
0
1-20
21-100
100
Largest cluster size
Negative
0.2 mm (pN0i)
0.3-2.0 mm (pN1mi)
2.0 mm (pN1a)

No.

%
57
24-83

123
245

33
67

211
157

57
43

319
49

87
13

58
134
110
49
17

16
36
30
13
5

339
29

92
8

318
50

86
14

64
180
123
1

17
49
34
1

285
83

77
23

285
50
33

77
14
9

285
60
23

77
17
6

285
23
17
43

77
6
5
12

285
61
17
5

77
17
5
1

Abbreviations: IHC, immunohistochemistry; H&E, hematoxylin-eosin.

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JOURNAL OF CLINICAL ONCOLOGY

Occult Axillary Node Metastases in Breast Cancer

Table 2. Number of Metastatic Cells and Largest Cluster Size, by Method of Staining
Pattern of Staining
IHC/H&E
Cells and Cluster Size
No. of metastatic cells
0
1-20
21-100
100
Largest cluster size
Negative (pN0)
0.2 mm (pNoi)
0.3-2.0 mm (pN0mi)
2.0 mm (pN1a)
Total

No.

IHC/H&E
%

285

No.

100

285

100

285

No.

44
32
24

1
1
31

98
2
0

12
16
5
33

22
16
12

IHC/H&E
%

Total

3
3
94

285
23
17
43

49
1
0
50

36
49
15

285
61
17
5
368

Abbreviations: IHC, immunohistochemistry; H&E, hematoxylin-eosin.

follow-up among survivors was 17.6 years (range, 3 to 25 years), and

was less than 5 years in only two patients. No patients were lost
to follow-up.
Detection of Axillary Node Metastases
A mean of 17 nodes (range, 5 to 43 nodes) were examined per
patient, and 23% of patients (83 of 368) were converted to node
positive. Among these 83, one positive node was found in 71% (59 of
83), two in 16% (13 of 83), three in 6% (five of 83), and more than
three in 6% (five of 83).
Among patients converted to node-positive, occult metastases
were first detected by H&E in 40% of cases (33 of 83), and by IHC in
60% (50 of 83; Table 1). The largest cluster size of the occult metastases
was no more than 0.2 mm (pn0i) in 73% (61 of 83), was 0.3 to 2.0
mm (pN1mi) in 20% (17 of 83), and was more than 2 mm (pN1a) in
6% (five of 83).
In 77% (63 of 83), the positive node/nodes were in level I. Metastases to level II with level I negative were found in 11% (nine of 83).
Because there were no metastases isolated to level III in the first 79
cases examined, level III nodes were not further examined in the
remaining patients.
Among patients converted to node-positive, 67% (56 of 83) had
invasive duct, 24% (20 of 83) had invasive lobular, 4% (three of 83)
had mixed ductal-lobular, and 5% (four of 83) had cancer of other
types (mucinous, tubular, papillary, medullary, and metaplastic).
Across the entire study, conversion to node positive was
noted in 20% of invasive duct (56 of 286), 41% of invasive
lobular (20 of 49), 33% of mixed duct-lobular (three of nine),
and 17% of other types (four of 24). Patients with invasive
lobular carcinoma comprised 13% of the study overall, 15% of
those with occult metastases detected by H&E, and 36% of those
with occult metastases detected by IHC.
Location and Extent of Nodal Metastases
Nodal metastases involved subcapsular sinuses in 48% (40
of 83) and lymph node parenchyma in 52% (43 of 83); cases
with both subcapsular and parenchymal metastases were designated as parenchymal. Metastases occurred as clusters in 67%
(56 of 83) and as single cells in 33% (27 of 83). Number of
www.jco.org

metastatic tumor clusters per patient ranged from one (14

cases) to more than 100 (four cases). Invasive lobular cancers
were over-represented among patients with single-cell (59%)
versus clustered metastases (7%).
In Table 2 we cross-tabulate number of metastatic cells and
largest cluster size by pattern of staining. Among the 50 patients who
were IHC/H&E, the number of metastatic cells varied widely, but
98% had a cluster size of no more than 0.2 mm (pN0i). For the 33
patients who were IHC/H&E, 94% had more than 100 cells
and only 15% (five of 33) had a cluster size of more than 2 mm
in size (pN1a).
In Table A1 we cross-tabulate number of metastatic cells by
cluster size, confirming that the greatest variation in number of metastatic cells was in patients with metastases of no more than 0.2 mm in
size, and that all patients with metastases more than 0.2 mm in size had
more than 100 cells.
Survival Analyses
Table 3 and Tables A2 and A3 (online only) report the results of
univariate analyses for OS, DFS, and DSD. Pattern of staining is
significant for all three outcome measures. Conversion to node positive, method of staining, number of positive nodes, number of metastatic cells, cluster size, tumor size, grade, and LVI are all highly
significant for DFS and DSD.
Table 4 and Tables A4 and A5 (online only) report the results of
multivariate analyses for OS, DFS, and DSD. Because pattern of staining, number of metastatic cells, number of positive nodes, and cluster
size all estimate lymph node tumor burden and are therefore correlated with each other, each of these variables is modeled separately
(models 1-4). All are highly significant for all three outcome measures, as are LVI, grade and tumor size for DFS and DSD.
OS, DFS, and DSD by method of staining are reported in Table
A6 (online only), in Figure 1, and in Figures A1 and A2 (online only).
OS, DFS, and DSD by cluster size are reported in Table A7 (online
only), in Figure 2, and in Figures A3 and A4 (online only). On the basis
of the relatively small number of total cases (368) and of patients
converted to node positive, (83) 95% CI are wide, but there is a clear
trend toward worse outcomes and especially disease-specific outcomes (DFS and DSD), with increasing lymph node tumor burden.
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Tan et al

Table 3. Univariate Analysis of DFS

Variable
Age, years
50
50
Tumor type
Ductal
Lobular
Grade
I
II
III
Lobular
LVI
Absent
Present
Tumor size, cm
1.0
1.1-2.0
2.0
Converted to node
positive
No
Yes
Pattern of staining
IHC/H&E
IHC/H&E
IHC/H&E
No. of nodes positive
0
1
1
No. of metastatic cells
0
1-20
21-100
100
Largest cluster size
Negative
0.2 mm
0.3-2.0 mm
2.0 mm

15-Year
DFS (%)

95% CI

RR

95% CI

73
78

64 to 80
72 to 83

1.0
0.8

0.5 to 1.2

.32

78
66

73 to 82
49 to 78

1.0
1.6

0.9 to 2.8

.08

95
69
76
66

85
60
67
49

98
77
83
78

1.0
6.5
5.1
7.6

2.0 to 21.2
1.5 to 16.7
2.2 to 25.9

.002

80
56

74 to 84
41 to 69

1.0
2.2

1.4 to 3.6

.001

87
78
68

76 to 93
71 to 84
59 to 76

1.0
1.7
2.7

0.8 to 3.7
1.3 to 5.9

.01

81
59

76 to 85
48 to 70

1.0
2.3

1.5 to 3.6

.001

81
66
49

76 to 85
50 to 78
31 to 65

1.0
1.9
3.1

1.1 to 3.3
1.8 to 5.4

.001

81
58
64

75 to 85
44 to 70
41 to 80

1.0
2.4
2.2

1.5 to 3.8
1.0 to 4.6

.001

81
60
82
50

76
34
55
35

to
to
to
to

86
78
94
65

1.0
2.0
1.0
3.1

0.9 to 4.2
0.3 to 3.2
1.9 to 5.2

.001

81
64
41
75

76
50
19
13

to
to
to
to

85
75
63
96

1.0
2.0
3.9
1.2

1.2 to 3.3
2.0 to 7.8
0.2 to 8.8

.001

to
to
to
to

Abbreviations: DFS, disease-free survival; RR, relative risk; LVI, lymphovascular invasion; IHC, immunohistochemistry; H&E, hematoxylin-eosin.

n 5.

DISCUSSION

In breast cancer, conventional histopathologic assessment of lymph

nodes consists of a single H&E-stained section per node. This technique, still endorsed by the College of American Pathologists as standard care,18 has long been recognized as flawed. Dowlatshahi et al19
have reviewed 25 separate articles spanning a 50-year period in which
negative axillary lymph nodes were re-sectioned to determine the
prognostic significance of missed nodal metastases. Although the investigators used various methods of serial sectioning and/or IHC
staining, 24 of 25 studies identified lymph node metastases in 7% to
42% of patients initially staged as node negative. Although many of the
studies were small and found no prognostic significance for lymph
1806

node micrometastases, six of the seven studies with more than 100
patients found DFS and/or OS to be significantly worse among patients with occult nodal disease. In the largest of these (n 921), the
Ludwig investigators used H&E-stained serial sections to identify occult metastases in 9% of cases and demonstrated significantly worse
DFS and OS at 5 years,20 and in a detailed reanalysis, at 10 years
as well.21
Unfortunately, the first Ludwig study required the examination
of about 1,600 slides to identify each additional node-positive patient,
an impossible workload for the routine examination of an ALND
specimen containing 15 to 20 nodes. With the advent of SLN biopsy,
and a pathologic specimen containing a median of two to three nodes,
enhanced pathologic examination became feasible for the first time
and seemed advantageous for a number of reasons. First, enhanced
pathology has allowed validation of the SLN hypothesis; in elegant
complementary studies, Turner6 demonstrated that a negative SLN is
highly predictive that the remaining nodes are negative, whereas
Weaver7 showed that the SLN is the node most likely to be positive.
Second, enhanced pathology seemed to increase staging accuracy by
identifying additional node-positive patients within each category of
tumor size.8 Third, Liberman9 showed that the false-negative rate of
SLN biopsy was significantly lower for those validation studies that
used IHC staining, compared with those which did not (8% v 3%,
respectively). Finally, enhanced pathology allowed more accurate prediction of non-SLN status10; for patients whose SLN were positive
only on IHC (compared with patients whose SLN was positive on
routine H&E), the chance of finding additional positive nodes on
completion ALND was substantially smaller. This information has
been incorporated into a multivariate nomogram by Van Zee et al,10
and has allowed a growing number of SLN-positive patients in our
own practice to avoid ALND altogether.22
Enhanced pathologic examination of SLN has also become controversial. In the absence of confirmatory level 1 evidence, are the
so-called advantages of pathologic upstaging and reduced falsenegative rate clinically relevant? Most importantly, are SLN micrometastases, especially those detected only by IHC or as isolated tumor
cells, prognostically significant? The present study addresses this issue
by drawing on a historic cohort of node-negative breast cancer patients: all had radical surgery, none had systemic adjuvant therapy, and
median follow-up was 17.6 years.
First, we demonstrate the feasibility of our pathologic technique.13,14 For patients with negative SLN frozen section, five additional slides are generated per SLN (one frozen section control, plus
one H&E and one IHC-stained section from each of two levels 50 m
apart). There is at present no consensus method of SLN examination,
and the spectrum ranges from routine single-section H&E (as recommended by the College of American Pathologists)18 to exhaustive
frozen section in which the SLN is examined in its entirety (entailing
30 or more sections).23 Our method represents a deliberate compromise between these two extremes, and has proven suitable for multiple
surgeons and pathologists in our high-volume practice.
Second, on serial sectioning/IHC, we found metastases in 23% of
patients (83 of 368) originally staged as node negative. Although 40%
of these were first seen on H&E, some were first identified by IHC and
confirmed by H&E after the fact. Even for those who would dispute
the clinical relevance of SLN metastases detected by IHC, the benefit of
IHC in helping to detect H&E-positive disease will be apparent to any
experienced pathologist and is indisputable.

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Occult Axillary Node Metastases in Breast Cancer

Table 4. Multivariate Analysis of Disease-Free Survival

Model 1
Variable

RR

Model 2

95% CI

Variable

Staining
IHC/H&E 1.0
IHC/H&E 1.5 0.9 to 2.7
IHC/H&E 3.3 1.8 to 6.0

No. of cells
.001
0
1-21
21-100
100
LVI
1.0
.002
Absent
2.3 1.4 to 3.8
Present
Grade
1.0
.008
I
6.7 2.0 to 22.0
II
5.2 1.5 to 17.9
III
8.4 2.4 to 29.9
Lobular
Tumor size,
cm
1.0
.01
1.0
0.9 0.4 to 2.0
1.1-2.0
1.8 0.8 to 3.9
2.0

LVI
Absent
Present
Grade
I
II
III
Lobular
Tumor size,
cm
1.0
1.1-2.0
2.0

RR

Model 3

95% CI

1.0
1.5 0.7 to 3.3
1.1 0.3 to 3.6
2.8 1.7 to 4.8

Variable

95% CI

Variable

RR

No. of nodes
.001
0
1.0
1
2.2 1.3 to 3.6
1
1.9 0.9 to 4.0

LVI
Absent
Present
Grade
1.0
.009
I
6.2 1.9 to 20.3
II
4.7 1.4 to 16.1
III
8.0 2.3 to 28.2
Lobular
Tumor size,
cm
1.0
.02
1.0
0.9 0.7 to 2.0
1.1-2.0
1.8 0.8 to 3.7
2.0

1.0
2.3 1.4 to 3.8

RR

Model 4

.002

Cluster size
.006
Negative
0.2 mm
0.3-2.0 mm
2.0 mm
LVI
1.0
.001
Absent
2.4 1.4 to 4.0
Present
Grade
1.0
.01
I
5.9 1.8 to 19.2
II
4.2 1.3 to 14.3
III
7.0 2.0 to 24.3
Lobular
Tumor size,
cm
1.0
.03
1.0
1.0 0.4 to 2.1
1.1-2.0
1.9 0.9 to 3.9
2.0

95% CI

1.0
.001
1.7 1.0 to 2.9
4.2 2.1 to 8.5
1.5 0.2 to 11.1
1.0
2.4 1.4 to 4.0

.001

1.0
6.1 1.9 to 20.1
4.8 1.4 to 16.1
7.7 2.2 to 27.3

.01

1.0
0.9 0.4 to 2.0
1.8 0.8 to 3.9

.01

Abbreviations: RR, relative risk; LVI, lymphovascular invasion; IHC, immunohistochemistry; H&E, hematoxylin-eosin.

Third, our data suggest that routine single-section H&E is adequate for the detection of lymph node macrometastases larger than 2
mm (pN1a disease). Among our 368 patients initially classified as
node negative, only 1% (five of 368) were found on re-examination to
have lymph node macrometastases (Table A1). Serial sectioning/IHC
is therefore of value primarily for the detection of micrometastases
(0.3 to 2 mm, pNmi) and submicrometastases ( 0.2 mm, pN0i).
Fourth, among patients converted to node positive, we identified
a single positive node in 71%. These are of course SLN, and this figure
closely matches our entire experience of SLN biopsy, in which approximately 70% of patients have had disease limited to the SLN.24 We also
identified skip metastases isolated to level II in 11% of node-positive

1.0

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1

Negative
0.2 mm
0.3 2.0 mm

0.9

Proportion Surviving

Proportion Surviving

1.0

-IHC/-H&E
+IHC/-H&E
+IHC/+H&E

0.9

cases. The term skip metastasis is a misnomer; since these represent

cases of direct lymphatic drainage from the breast to axillary level II,
skip metastases are, in reality, SLN.
Fifth, we identify a higher rate of conversion to nodepositive for invasive lobular than for invasive duct carcinomas
(40% v 20%), over-representation of invasive lobular among
IHC-detected versus H&E-detected disease (36% v 15%), and
over-representation of invasive lobular among patients with
single-cell versus clustered metastases (59% v 7%). All are consistent with the loss of e-cadherin expression characteristic of invasive
lobular carcinomas,25,26 and suggest the particular importance of
IHC staining for this distinctive entity.

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1

10

15

20

25

Time (years)
No. at risk (# events)
-IHC/-H&E
+IHC/-H&E
+IHC/+H&E

241 (35)
38 (11)
21 (11)

199 (48)
29 (14)
14 (16)

140 (51)
24 (16)
9 (16)

15

20

25

Time (years)
32 (53)
8 (16)
0 (16)

Fig 1. Disease-free survival by pattern of lymph node staining, IHC/H&E (n

285) versus IHC/H&E (n 50) versus IHC/H&E (n 33); P .001. IHC,
immunohistochemistry; H&E, hematoxylin-eosin.
www.jco.org

10

No. at risk (# events)

Negative
0.2 mm
0.3 2.0 mm

241 (35)
46 (14)
10 (7)

199 (48)
34 (19)
7 (10)

140 (51)
28 (21)
4 (10)

32 (53)
8 (21)
0 (10)

Fig 2. Disease-free survival by largest cluster size, negative (pN0, n 285)

versus 0.2 mm (pN0i, n 61) versus 0.3 to 2.0 mm (pN1mi, n 17); P .001.
Results for cluster size more than 2.0 mm (pN1a, n 5) are not shown.
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Tan et al

Sixth, we examine the site and distribution of nodal metastases.

While the site of metastasis was equally likely to be subcapsular or
parenchymal, the extent of metastasis varied widely, ranging from one
to more than 100 clusters. Among IHC/H&E patients, the number
of metastatic cells varied widely, but 98% had pN0i disease, with a
cluster size of no more than 0.2 mm (Table 2). The greatest variation in
number of metastatic cells was in patients with no more than 0.2 mm
of disease (Table 3), suggesting biologic heterogeneity even within the
smallest subcategory of cluster size. Although these observations in
general support the most recent AJCC Staging Manual (ed 6),15 in
which nodal metastases are categorized by cluster size, they also illustrate the difficulty of fitting a heterogeneous collection of pathologic
findings into a single system of classification, particularly for patients
with invasive lobular cancers.
Finally, we demonstrate that lymph node micrometastases
missed by conventional single-section H&E and detected by our current pathologic protocol for SLN are prognostically significant. These
findings are particularly striking for the disease-specific outcome measures DFS and DSD, where every measure of lymph node tumor
burden (pattern of staining, number of positive nodes, number of
cells, and cluster size) was highly significant on univariate analysis
(Tables 3 and A3), and on multivariate analysis retained a significance
comparable to, or greater than, tumor size, grade, and LVI (Tables 4
and A5). The prognosis of patients with micrometastases detected by IHC was worse, and the prognosis of those detected by
H&E was substantially worse, than that of patients who remained
node negative. These differences were most apparent at 10 or more
years of follow-up.
Our study has some limitations. The design is retrospective. The
primary tumor slides were missing in 20% (and the axillary tissue
blocks in 30%) of eligible patients. The breast cancers of the 1970s
were largely symptomatic, while present-day lesions are increasingly
screen-detected at an earlier stage. Despite excellent long-term followup, our results are limited by relatively small numbers of node-positive
patients (50 detected by H&E and 33 by IHC). The data reflect our
own pathology protocol, and may not apply for institutions which use
different methods. Finally, none of our patients received systemic therapy.
Our data nevertheless clearly establish the value of our current
SLN pathology protocol (serial sections and IHC staining) for reexamining archival material for lymph node metastases missed by conventional single-section H&E, and demonstrate the prognostic
significance of those metastases, whether detected by H&E or IHC. We
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AUTHORS DISCLOSURES OF POTENTIAL CONFLICTS
OF INTEREST
The author(s) indicated no potential conflicts of interest.

AUTHOR CONTRIBUTIONS
Conception and design: Lee K. Tan, Dilip Giri, Amanda J. Hummer,
Katherine S. Panageas, Patrick I. Borgen, Hiram S. Cody III
Financial support: Larry Norton, Clifford Hudis, Patrick I. Borgen,
Hiram S. Cody III
Administrative support: Lee K. Tan, Edi Brogi, Larry Norton, Clifford
Hudis, Patrick I. Borgen, Hiram S. Cody III
Provision of study materials or patients: Lee K. Tan, Edi Brogi
Collection and assembly of data: Lee K. Tan, Dilip Giri, Amanda J.
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Hiram S. Cody III
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Appendix
The Appendix is included in the full-text version of this article, available online at www.jco.org. It is not included in the PDF version
(via Adobe Reader).

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1809