Intraoperative lumpectomy margins assessment in patients with early-stage
breast cancer treated with breast conservation therapy: frozen section
analysis versus imprint cytology
Mostafa El-Sayed1, Gamal Saleh1, Ahmed Nada2 , Nashwa Omara*& Hussam Hussein**
Departments of General Surgery1 & Pathology* Benha University Hospital- Departments of
General Surgery2 & Pathology** Cairo University Hospital, Egypt
ABSTRACT
This cross-sectional comparative randomised study was designed to evaluate the accuracy of
Intraoperative lumpectomy margins assessment in patients with early-stage breast cancer treated with
Breast-conserving therapy ; frozen section analysis versus imprint cytology. The study comprised 40
female patients with mean age of 47.1±5.5. The patients were randomized into 2 equal groups: frozen
section group & imprint group. After adequate margins had been achieved, additional 5 mm normal
breast tissues were removed all around the wound site and subjected to paraffin section examination.
There was a non-significant difference in both groups as regards the need of intraoperative reexcision. The mean operative time was significantly longer in frozen section group (105.4± 17.4
minutes) compared to that recorded in imprint group (85.1±16.2 minutes). On paraffin section
examination, there was a significant higher rate of positive margin in frozen section group. The
accuracy rate of frozen section analysis and imprint cytology to define positive margin was 85% &
100% respectively. Both techniques were effective in reducing the need of a second operation for
margin control. However, imprint cytology; in addition to saving tissue for paraffin histopathological
examination; has the advantages of being more accurate to ensure clear margins with significant
decrease in the operative time.
Keywords: Breast-conserving therapy, frozen section, imprint cytology.
INTRODUCTION
Breast-conserving therapy (BCT) has gained wide acceptance as providing long-term survival
equal to that seen with mastectomy for early-stage breast cancers, and accordingly the
number of lumpectomy procedures has increased dramatically(1).
The goal of BCT should be to remove the smallest amount of tissue possible but still remove
the tumor with adequate negative margins. Although these patients will receive radiation
therapy to the preserved breast, radiation cannot completely compensate for inadequate
surgery. The appropriate margin width is debated in many literatures. Inadequate surgical
margins represent a high risk for adverse clinical outcome in BCT. The majority of studies
report unacceptably high local recurrence rates when tumor cells are present at the cut surface
of the specimen. Moreover, positive resection margins in 20% to 40% of the patients who
underwent BCT had been reported. This may result in an increased local recurrence rate or
additional surgery and, consequently, adverse affects on cosmoses, psychological distress,
and health costs(2-3).
The first operation provides the best opportunity to achieve an acceptable cosmetic outcome
over subsequent operations to clear positive margins, thereby establishing the need to
accurately assess the margin status intraoperatively. Frozen section has been the traditional
method of microscopic analysis of margins and is widely used at many institutions for
oncologic procedures. Because of the relative ease and the wide experience gained, this
technique has been applied frequently to assess tumor margins during lumpectomy. The
excised specimen is frozen, sliced, and analyzed microscopically. The use of frozen section
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unfortunately causes permanent loss of tissue, sampling errors, and histologic artifacts related
to tissue preparation (4-6).
Intraoperative touch preparation cytology (IOTPC) or ‘‘imprint cytology’’ is a promising
alternative to frozen section analysis (FSA). The technique is based on the histological
characteristics of the cell surface of malignant cells, which stick to glass surfaces, whereas
benign mammary fat tissue does not. To assess margin status, a glass slide is brought against
the borders of the excised specimen. Next, cells sticking to the glass surface are fixated,
stained, and microscopically evaluated. Several studies have concluded that IOTPC is
inexpensive, accurate, quick, and saves tissue for permanent sectioning and histopathological
examination(7-10).
This study was designed to evaluate the accuracy of Intraoperative lumpectomy margins
assessment in patients with early-stage breast cancer treated with BCT; frozen section
analysis versus imprint cytology.
PATIENTS & METHODS
This cross-sectional comparative randomised study was conducted at General Surgery in
conjunction with Pathology Departments, Benha & Cairo University Hospitals over a period of
3 years, started April 2007 and comprised 40 female patients with mean age of 47.1±5.5; range
37-56 years. All patients underwent full clinical examination, preoperative mammography, and
preoperative fine needle aspiration cytology. They were assigned for BCT according to
indicated operative procedure as described by Dixon(11).
With Ethics Committee approval, all patients were informed and consented before surgery
after explanation & discussion of the procedure and possible complications of various surgical
modalities for treatment of breast cancer.
Through the surgical procedure lumpectomy was performed with 1 cm margin of surrounding
normal breast tissue, (Fig. 1). Complete axillary evacuation up to level III was carried on with
preservation of the thoracodorsal and long thoracic nerves (Fig. 2-4), then wounds were closed
after axillary drainage, (Fig. 5).
The patients were randomized into 2 groups:
 Frozen section group (n=20 patients) assigned to undergo intraoperative assessment of
surgical margins of lumpectomy specimen using FSA.
 Imprint group (n= 20 patients) assigned to undergo intraoperative assessment of surgical
margins of lumpectomy specimen using IOTPC.
The lumpectomy specimens were oriented as superior, medial, lateral, inferior, deep and
superficial using suture material and all the 6 margins were evaluated.
Procedure used in intraoperative frozen section analysis:
 The specimen was sliced into 4–5 mm thick sections perpendicular to the longest axes of
tumor mass and mounted on a cryrostat and placed on slides and stained with hematoxylin
and eosin. After preparation, the slides were examined by the pathologist, and histological
2
tumor type, maximal dimension of invasive carcinoma, and closest distance in millimeters
between inked surgical margins and invasive carcinoma were reported , (Fig. 6).
 Negative surgical margin was defined as tumor cells were > 2 mm from the inked surface of
the lumpectomy specimen. Tumor positive margin was defined as tumor cells were present
at the inked edge of specimen. Close margin was defined as tumor cells were ≤ 2 mm from
the inked surface(12).
Procedure used in intraoperative imprint cytology:
The slides were air dried and stained immediately with the Diff-Quik stain as follows:
 The slides were fixed for 15–20 seconds in solution 1. Excess solution was drained onto a
paper towel.
 The slides were dipped repeatedly for 15–20 seconds in solution 2 until slides were
uniformly coated and turned reddish-pink. Excess stain was drained.
 The slides were dipped repeatedly for 15–20 seconds in solution 3. Rinsed in tap water.
Excess water was drained.
 The slide smears were examined for quality of stain (purple). If the stain was too pale or too
intense (deep blue), it may be re-stained by repeated immersion in solution I for 20–30
seconds.
 Intraoperative/stat reading may be performed on uncoverslipped wet slides. Mount in resin
and coverslip slides when dry(5) , (Fig. 7) .
If the margins were found to be positive another 1cm safety margin was excised from the side
of the cavity that corresponded to the positive margin, and the outer surface of re-excised
specimen was marked by sutures. Histopathological re-evaluation was done. If positive
margins were insisted and clear margins cannot be obtained with two re-excisions, we
performed a mastectomy and the patient was excluded off the study.
After adequate margins had been achieved, additional 5 mm normal breast tissues were
removed all around the wound site and subjected to paraffin section examination by two
pathologists, (Fig. 8).
The operative time was calculated on the basis of the surgical procedure added to time taken to
perform imprint cytology or frozen section procedure.
Statistical analysis
The collected data were tabulated and analyzed using t-test and Z-test. Statistical analysis was
conducted using the SPSS (Version 16) for Windows statistical package. Values of P <0.05
were considered significant.
3
Fig 1. lumpectomy of the tumor with safety
margin & axillary incision.
Fig 2. Dissection of the axillary vein (v).
Fig 3. Axillary clearance showing intercostobrachial nerve (I), long thoracic (LT) &
Thoracodorsal (TD) nerves.
Fig 4. Axillary Dissection Completed.
Fig 5. Both lumpectomy and axillary wounds were
closed with axillary drainage.
Fig 6. Frozen section of the surgical margins of
lumpectomy specimen showing malignant cells (arrow).
4
Fig 7. Imprint cytology of the surgical margins of
lumpectomy specimen showing malignant cells
(arrow).
Fig 8. 5mm normal breast tissues were removed all
around the wound site for paraffin section examination.
RESULTS
This study comprised 40 female patients with mean age of 47.1±5.5; range 37-56 years.
There was a non-significant difference between patients enrolled in both groups as regards
the age. There were 16 patients with lesion diameter ≤2 cm, with no clinically palpable
axillary lymph nodes, and 24 patients with lesion diameter ranging between >2 and ≤4 cm; 3
of them had no clinically palpable lymph nodes and 21 had palpable lymph nodes (N1). There
was a non-significant difference between patients enrolled in both groups as regards the
tumor size or clinical nodal status, Table 1.
There were 5 patients in frozen section group required intra-operative re-excision because of
positive margin; 4 (20.0%) required only one re-excision and the other patient (5.0%)
required two re-excisions. In imprint group, 6 patients required intra-operative re-excision; 5
(25.0%) required one re-excision and the other patient (5.0%) required two re-excisions; with
non-significant difference in both groups as regards the need of intra-operative re-excision.
The median duration of frozen section and imprint cytology procedure was 28.07 and 15.25
minutes respectively. The mean operative time was 105.4± 17.4 ; range 85-112 minutes in
frozen section group and 85.1±16.2; range 64-95 minutes in imprint group with a
significantly (P<0.05) shorter operative time in imprint group , Table 2.
All patients were discharged 24 hours after surgery. Drains were removed when the drainage
was less than 30 ml a day. Paraffin section examination of the excised 5mm breast tissue
around the specimen showed negative margins in all cases of imprint group. Whereas,
positive margins were observed in three cases in frozen section group with more than 2mm
free margin. There was a significant higher rate of positive margin in frozen section group.
The accuracy rate of FSA and IOTPC to define positive margin was 85% & 100 %
respectively. No cases in either group required reoperation because, on paraffin section
examination of the excised 5mm breast tissue around the specimen, all cases had > 2mm free
margin.
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Table 1- Patients clinical data.
Frozen section
group
N=20
47.4±6.1
Age (years) *
Clinical tumor size (cm)
Clinical nodal status
≤2
>2-≤4
N0
N1
7
13
10
10
Imprint
group
Total
N=20
46.9±
47.1±5.5
5
9
16
11
24
9
19
11
21
Test of
significance
P
t= 0.28
>0.05
Z=0.50
Z=0.41
Z=0.23
Z=0.22
>0.05
>0.05
>0.05
>0.05
Mean (X-) ±SD *
Table 2- Patients variables of the study groups.
Operative time (min)*
No. having 1 intra-operative re-excision
No. having 2 intra-operative re-excision
Positive margin by paraffin:
> 2mm free margin
≤ 2mm free margin
Reoperation
Frozen section
group
N=20
105.4± 17.4
4(20.0%)
1(5.0%)
3(15.0%)
0(0.0%)
0(0.0%)
Imprint group
N=20
Test of
significance
P
85.1±16.2
5(25.0%)
1(5.0%)
t=3.82
Z=0.33
-
<0.05
>0.05
-
0(0.0%)
0(0.0%)
0(0.0%)
Z=1.80
-
<0.05
-
Mean (X-) ±SD *
DISCUSSION
During the last 20 years, BCT has come to the forefront of treatment options for breast cancer
with equivalent survival rates to those obtained after mastectomy (13, 14). The success of BCT
depends not only on appropriate patient selection but also on adequate surgical margins
combined with a cosmetically pleasing result(7).
Precise preoperative assessment of the extent of breast cancer is one of the important points
for successful local control in breast conserving surgery. The newer diagnostic imaging
techniques including magnetic resonance imaging, breast tomosynthesis, and
fluorodeoxyglucose-positron emission tomography /computed tomography may be used for
more accurate evaluation of disease extent and multifoci, so they may help the selection of
the patients for breast conserving surgery and extent of the surgical resection (15). But further
studies are warranted to adapt these new imaging methods to achieve tumor-free margins
during breast conserving surgery(12).
The definition of negative or close margins varies widely among authors, so optimal width of
excision is not clear. Because the lack of uniformity in margin definition, local recurrence
and survival results of BCT couldn’t be compared adequately in literature. But various
studies have showed that involved or close margins are considered the strongest predictor for
local failure. Radiotherapy alone cannot compensate for inadequate surgery(2,16-19).
Skripenova & Layfield(20) reported that optimal surgical technique requires microscopic
margins free of carcinoma by at least 2 mm .
Many authors performed excision biopsy or lumpectomy without margin assessment in initial
operation, and further surgery are required if positive margins exist in permanent sections.
However all authors accepted that tumor recurrence rate are extremely higher in patients who
have tumor cells on the cutting surface of specimen. Moreover, repeated operations may
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cause many negative results such as poor cosmetic appearance as it is well known that the
best cosmetic result after breast conservation therapy occurs after only a single excisional
biopsy is performed, anesthesia risks, adverse psychological reactions, delay on starting
oncological treatments and higher cost(18,21,22).
All of these problems can be prevented by intraoperative margin assessment and repeated reexcisions can be made in a single operation if necessary. Moreover, series which were
performed intraoperative margin analysis have lower recurrence and mortality rates than
conventional histological margin analysis(4,7,23). Numerous methodologies have been
proposed for intraoperative evaluation, and there is no standardized method(21).
In our study intraoperative lumpectomy margins assessment were randomly done using either
FSA or IOTPC. This agreed with Dener etal., (12) who reported that these techniques are the
best methods for intraoperative evaluation of lumpectomy margins. Also Mannell(24)
observed that when there is a feedback between the surgeon and pathologist during the
operation and margins are judged clear of the cancer , only a small number will require reexcision. Eleven patients; 5 (25%) in frozen section group and 6 (30%) in imprint group had
intraoperative positive margin and benefiting from immediate intervention in this study with
no need for reoperation.
Because of permanent tissue loss after freezing, additional 5 mm normal breast tissues were
removed all around the wound site and subjected to paraffin section examination. This small
part of tissues did not interfere with cosmoses. This agreed with Cox etal.,(4) who observed
that the first operation provides the best opportunity to achieve an acceptable cosmetic
outcome over subsequent operations to clear positive margins.
Our results were comparable with other studies which retrospectively analyzed the influence
of FSA on BCT outcome and found similar figures (24% to 27%) of the patients underwent
additional tissue excision based on FSA, whereas 5% to 9% required a second re-excision
procedure. The Accuracy of FSA was 84% when evaluated on a per-case basis, and 96% on a
per-slide basis(6,25-27). However in our study on definitive histopathological examination 15%
of patients had positive margin ( malignant cells were found at the inner border of the
additional 5 mm normal breast tissue in 3 patients with free margin > 2mm) with 85%
accuracy & no case required re-operation. This could be attributed to the small number of
patients in our study or the difference in experience of the pathologists.
In this study, imprint cytology had a diagnostic accuracy of 100% when compared with the
final examination of margins in paraffin sections. These results go in hand with that reported
by Klimberg et al.,(8) And Ku & Cox(28). However, this is better than 94% reported by
Mannell(24). The more accuracy of imprint cytology than frozen section examination could be
attributed to the possibility to survey the entire surface area of the lumpectomy margin using
imprint cytology, however as such survey is not practical with frozen section technique (12, 21).
In the current study, the mean operative time was significantly longer in frozen section group
(105.4± 17.4 ; range 85-112 ) compared to that recorded in imprint group (85.1±16.2; range
64-95 minutes), reflecting the technical demands and the prolonged duration required for
FSA.
It could be concluded that intraoperative margin assessment by FSA or IOTPC is an effective
procedure in reducing the need of a second operation for margin control. However, imprint
7
cytology, in addition to saving tissue for paraffin histopathological examination, has the
advantages of being more accurate to ensure clear margins with significant decrease in the
operative time.
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