Review Article
Examination of Surgical Specimens of the Esophagus
Pablo A. Bejarano, MD; Mariana Berho, MD
 Context.Esophageal cancer continues to be one of the
most lethal of all gastrointestinal malignancies. Its prog-
nostic parameters are based on the gross and histopatho-
logic examination of resected specimens by pathologists.
Objective.To describe the implications of appropriate
handling and examination of endomucosal resection and
esophagectomy specimens from patients with esophageal
carcinoma while considering the implications of the
surgical techniques used to obtain such specimens.
Parameters include histopathologic findings necessary for
accurate staging, differences in the assessment of margins,
residual malignancy, and criteria to evaluate for tumor
regression after chemoradiation therapy as well as the role
E sophageal carcinoma is one of the most lethal of all of
the gastrointestinal malignancies. The most frequent
esophageal malignancy is adenocarcinoma, which continues
to be diagnosed at a rate of 5% to 10% per year in some
Western populations, in part because of the rise of Barrett
esophagus and the epidemic of obesity.1 Squamous cell
carcinoma (SCC) is more frequent in China, Iran, and some
African countries, and is associated with the use of tobacco,
the use of alcohol, and the ingestion of burning-hot drinks.
The pathologists role in examining esophageal surgical
specimens is to determine the parameters that mark the
probabilities of patient survival, and thus guide the
therapeutic approaches. Accurate histologic evaluation
may be dependent on adequate handling of the gross
specimen. The specimens received in the surgical pathology
laboratory include endoscopic mucosal resections (EMRs)
and esophagectomies.
The purpose of this review is to describe the handling and
approach in examining surgically obtained specimens that
contain carcinoma that may or may not have been treated
with neoadjuvant therapy. Both adenocarcinoma and SCC
will be similarly analyzed.
ENDOMUCOSAL RESECTION
Endomucosal resection offers a less extensive surgical
therapy to a select group of early-stage esophageal
carcinoma patients by endoscopically removing the tumor
Accepted for publication January 26, 2015.
From the Department of Pathology, Cleveland Clinic Florida,
Weston.
The authors have no relevant financial interest in the products or
companies described in this article.
Reprints: Pablo A. Bejarano, MD, Department of Pathology,
Cleveland Clinic Florida, 3100 Weston Rd, Weston, FL 33331 (e-
mail: bejarap@ccf.org).
1446 Arch Pathol Lab MedVol 139, November 2015
of immunohistochemistry and the judicious use of frozen
sections.
Data Sources.Sources were a review of the literature
and the authors experience handling these types of
specimens.
Conclusions.Examining surgical specimens of the
esophagus is critical in the management of patients with
esophageal carcinoma, and it requires careful consider-
ation of the diagnostic pitfalls, staging-related parameters,
and results of molecular tests.
(Arch Pathol Lab Med. 2015;139:14461454; doi:
10.5858/arpa.2014-0506-RA)
and a rim of margin tissue. It should be considered the
therapy of choice when dysplasia shows a visible lesion and
for pT1a (intramucosal) adenocarcinoma. It is important to
note that preoperative evaluation should be as accurate as
possible because a higher pathologic stage will preclude a
successful complete removal of the tumor by this method.
Thus, only tumors that are limited to the mucosa or
submucosa are suitable for EMR.2,3 Endomucosal resection
is also an alternative to surgery for treatment of T1b
adenocarcinomas limited to the upper aspect of the
submucosa and without lymphovascular invasion. However,
EMR is not a curative procedure for deeper tumors involving
a deeper aspect of the submucosa or the muscle.4
EMR Technique
Endomucosal resection is performed using either the cap-
assisted technique attached to the tip of the endoscope or
the rubber band technique. The area to be removed may be
preinjected and lifted with a solution of saline methylene
blue and epinephrine. The area of resection is suctioned into
the cap and then grasped with a monofilament snare for
resection. For the rubber band technique, the tissue
suctioned into the cap is grasped by deploying a rubber
band forming a pseudopolyp. The banded pseudopolyp
containing the tumor is then cut with a snare at its base,
ensuring that some of the uninvolved surrounding mucosa
and/or submucosa is attached to the tumor. The British
Gastroenterology Association recently stated that the cap
and snare technique with submucosal injection and the
band ligation technique without submucosal injection are
considered equally effective by experienced endoscopists in
high-volume tertiary care centers.5,6
Gross Pathology
The EMR specimen may have a round or oval shape, and
it may be oriented with a stitch to mark 12 oclock. The base
Examination of Surgical Specimens of the EsophagusBejarano & Berho
and the mucosal margins are inked and the specimen may
be pinned on a rigid support, such as a corkboard, and
should be allowed to fix in formaldehyde for 12 to 24 hours.
Photographs of the specimen may be particularly useful
when the case is to be discussed in a multidisciplinary
conference. It should be serially sectioned at 2-mm intervals
and completely embedded. The margins can be taken en
face, unless a gross lesion is located at less than 1 mm from
the margin. In the latter case, perpendicular sections can
include both the lesion and margins. It is critical that the en
face section be of full thickness including mucosa, submu-
cosa, and muscularis, because carcinoma may involve any of
these layers. The overlying mucosa may curl over the edge
of the cut margin. Therefore, it may be necessary to gently
pull back the mucosa to line it up over the muscularis before
taking the section. The sections of the margins should be
taken from the area closest to the site of the tumor.
Histology
Meticulous handling of the specimen is important because
assessment of the margins is crucial to determine the
success of the surgical procedure, since the presence of
tumor cells at the margins indicates an incomplete resection
warranting further treatment. Although only the presence of
carcinoma cells at the inked or cauterized margins is
considered positive, it is appropriate to provide a measure-
ment of distance between the tumor and the closest margin
in all cases. This goal can be accomplished when a single
complete specimen is obtained. If the procedure yields
multiple fragments, evaluation of the side margins is
hampered and may only allow for an accurate evaluation
of the deep margin.
Neoplastic changes are graded according to criteria for
dysplasia and carcinoma in Barrett esophagus.7 Low-grade
dysplasia is based more on cytologic changes with minimal
to absent architectural abnormalities. The glands and
surface epithelium are lined by elongated, hyperchromatic,
and pseudostratified nuclei. High-grade dysplasia (HGD)
has more pronounced architectural abnormalities, including
crypt branching. There is nuclear stratification, increased
nucleus to cytoplasmic ratio, loss of nuclear polarity, and
frequent mitoses. Intramucosal carcinoma (IMC) is charac-
terized by the merging of abnormal glands expanding in the
lamina propria and penetrating through the basement
membrane into the lamina propria and muscularis mucosae,
but not beyond the muscularis mucosae. Invasive carcinoma
shows invasion through the muscularis mucosae into the
submucosa and beyond.
In addition to meticulous handling, it is equally important
that the pathologist stages (pT) the tumor. In some centers,
stage T1b defined as submucosal involvement is further
subclassified as sm1 if there is invasion of the upper third of
the submucosa up to less than 500 microns, sm2 for
invasion of the middle third, and sm3 when there is invasion
of the lower third. Unfortunately, the muscularis propria is
not present in most cases, and thus determining where the
submucosa starts and ends can be difficult to establish.
Alternatively, it is appropriate to measure the depth of
submucosal invasion with a micrometer. Likewise, measur-
ing the distance between tumor and the deepest resection
margin is recommended. Tumor cells at that deeper margin
indicate that the tumor was not completely excised, a setting
which may warrant further treatment.
It is pivotal not to confuse muscularis mucosa with
muscularis propria. The former varies in organization, from
Arch Pathol Lab MedVol 139, November 2015 Examination of Surgical Specimens of the EsophagusBejarano & Berho 1447
sparse bundles of smooth muscle in the upper esophagus to
a thickened network in the distal portions.8 In fact, the
muscularis mucosa in Barrett esophagus is characteristically
duplicated, and the spaces between the duplicated fibers of
smooth muscle may be erroneously interpreted as submu-
cosa, leading to overstaging in the presence of tumor cells.9
The presence of tumor between the duplicated muscularis
mucosae has a similar low risk of lymph node metastases to
involvement of lamina propria if lymphovascular invasion is
absent.3,4 The duplication of the muscularis mucosa adds
even more complexity to accurate interpretation, as attested
by the fact that the diagnosis of IMC has low interobserver
agreement.10,11 Separation between HGD and IMC may
dictate the therapeutic approach. However, pathologists
have only moderate interobserver agreement in the
separation of HGD from IMC. As mentioned above, in
IMC the neoplastic cells penetrate through the basement
membrane, but recognizing this feature is sometimes
difficult. Criteria for IMC include anastomosing gland
pattern in what is known as back-to-back architectural
glandular arrangement that cannot be readily explained by
the presence of involvement of preexisting crypts,12 as well
as the presence of sheetlike growth pattern or the presence
of single-cell invasion (Figures 1 and 2).
The presence or absence of lymphovascular space
invasion, intestinal metaplasia, and overlying dysplasia
should be reported. Although a given specimen may show
only dysplasia without carcinoma, the endoscopist may
appreciate a comment in the pathology report as to which
layers of the esophagus are histologically present in the
sample. Such feedback may allow the endoscopist to learn
how deep the sample is by mentioning that it contains
mucosa, submucosa, muscularis propria, and perhaps
deeper tissue, if present.
Immunohistochemistry
The use of immunohistochemistry in the evaluation of
dysplasia/carcinoma of the esophagus is limited. Because
stain for p53 may distinguish reactive and regenerative
changes from true dysplasia, its value is mainly in biopsy
specimens prior to a definitive therapy. The use of p53 on
resection specimens can be applicable when one sees
atypical cells at the mucosal margin, where it is difficult to
decide whether the atypia is indeed reactive or dysplastic.
Extrapolating the use of p53 in biopsy tissues, a positive p53
in those cells present at the margin would strengthen the
support for dysplasia.13,14 There is no practical value for the
use of CK7 and CK20 stains in EMR specimens. Because of
the high stakes of a positive margin for dysplasia or
carcinoma, an opinion from a second pathologist may be
warranted. If Her2 (Human epidermal growth factor-2)
immunostaining or fluorescent in situ hybridization (FISH)
has not been performed on the preoperative biopsy tissue,
either test should be requested at this time. Testing for Her2
is discussed later in this review.
Artifacts
Endoscopic resection specimens may show a number of
artifacts, such as hemorrhage, cautery distortion, loss of the
surface epithelium, and fibrin deposition, that may occur at
the time of suction. Also, the fixation in formaldehyde
contracts the tissues, and the edges of the specimen may
fold in on themselves. These artifacts may make appropriate
orientation of the specimen by the histotechnologists
difficult, and thus hinder the histologic assessment by the

Figure 1. Distinction between high-grade dysplasia (HGD) and
intramucosal carcinoma (IMC). A, Dysplastic cells are limited to the
surface epithelium and the crypt profiles, with no stromal involvement
in HGD. B, Individual cells in the stroma (arrows) of lamina propria is a
feature that distinguishes IMC from HGD (hematoxylin-eosin, original
magnification 3100).
Figure 2. Intramucosal carcinoma (IMC) at the gastroesophageal
junction. Back-to-back glandular arrangement (arrows) that cannot be
readily explained by the presence of involvement of preexisting crypts is
also a feature of IMC (hematoxylin-eosin, original magnification 3100).
1448 Arch Pathol Lab MedVol 139, November 2015
pathologist as well. One must also be aware that entrapped
glands and submucosal glands may mimic submucosal
adenocarcinoma.
Follow-Up
The pathologist needs to be informed of previous EMR at
the time of review of follow-up biopsy tissues in order to
avoid potential pitfalls, such as the so-called buried
dysplasia, where groups of atypical glands are under the
re-epithelized surface squamous mucosa. These foci may be
confused with invasive carcinoma. Moreover, biopsy tissues
obtained 1 to 16 days after endoscopic resections can show
signet ring cell change and clear cell degeneration in areas
of ischemia, which also can be mistakenly diagnosed as
malignancy.15
ESOPHAGECTOMY SPECIMENS
Most of the surgically obtained esophagi that harbor
adenocarcinoma or SCC are submitted to the pathologist
for accurate staging, assessment of the resection margins,
and other features that also play a role in the prognosis. In
cases of patients who had undergone neoadjuvant therapy,
the pathologists role is expanded to determine the amount
of residual tumor burden or tumor regression. Occasion-
ally, esophagectomies are performed as the only viable
therapeutic approach for other types of neoplasias,
including primary malignant melanomas, gastrointestinal
stromal tumors, or leiomyomas, which can be multiple or
very large.
Intraoperative Consultation (Frozen Section)
Very often, the pathologists initial evaluation of the
esophagectomy specimen occurs while the patient is still
under anesthesia. The decision to reconstruct and anasto-
mose may be decided by the surgeon based on the gross
and/or microscopic tumor status of the margins. To enable
this critical intraoperative decision, the pathologist may
receive separate rims of tissue corresponding to either the
proximal or distal margins, or both, for frozen section
examination. The distal margin is usually a portion of the
stomach. Alternatively, the pathologist may have to examine
the entire specimen grossly prior to submitting the margins
for microscopic examination. To do so, the pathologist will
measure the distance between the tumor and the proximal
and distal margins. In most cases, frozen section slides are
prepared for interpretation and reported within 20 min-
utes.16 Because adenocarcinomas often grow underneath the
uninvolved mucosa, it is very important that when
evaluating the proximal and distal margins, a full thickness
of the esophageal or gastric wall be taken and not just the
mucosa. As mentioned above, for EMRs the luminal
margins can be taken en face. If the gross lesion is less
than 1 cm from the margin, perpendicular sections that
include both the lesion and margin are preferable. The
overlying mucosa may curl over the edge of the cut margin.
It may be necessary to gently pull back this mucosa to line it
up over the muscularis before taking the section. The
margin section should be taken from the area closest to the
tumor.
If the pathologist sees malignant cells at the margins, the
surgeon may resect an additional rim from the ends of the
esophagus or stomach for a new evaluation by means of
frozen section. The deep/circumferential margin may need
to be evaluated; however, the area should be painted with
Examination of Surgical Specimens of the EsophagusBejarano & Berho
ink before it is cut into. Once inked, the lumen of the
esophagus can be probed with a finger to detect the location
of the tumor. This probing is helpful when longitudinally
opening the esophagus along the wall opposite the tumor,
to avoid cutting into and/or distorting the tumor. With the
esophageal lumen and tumor completely exposed, the
pathologist proceeds to cut a full-thickness slice in the
middle of the tumor to observe the closest point of the inked
deep margin to the tumor and perform a frozen section of
the slice.
Gross Examination
The next step is to properly fix the esophagus for 24 hours
in order to better obtain the necessary oncologic informa-
tion on the permanent tissue sections. Frozen sections or
intraoperative examination may not have been performed.
Because fixation in formaldehyde may cause marked
irregular and distorted contraction of the specimen, it is
preferable to extend the entire esophagus specimen and pin
it to a corkboard or Styrofoam. In addition, it should be
turned over so that the mucosa will face down on the
formaldehyde liquid. A piece of paper towel between the
corkboard and the specimen helps formalin to penetrate
into the adventitia/deep aspect of the specimen. One should
expect shrinkage of about 10% after the specimen has been
fixed in formaldehyde for 24 hours, and as such, it is
preferable to obtain pertinent measurements as soon as
possible. As with the EMR specimens, a photograph may
prove useful when analyzing the case with clinical
colleagues.
Key characteristics of the tumor should be noted,
including size, color, and whether it is exophytic (polypoid),
ulcerated, or infiltrative, as well as the distance to the
gastroesophageal junction (GEJ) and to the margins. Depth
of invasion identifying the wall layers of the esophagus, the
percentage of circumference involved, and dilation of the
organ proximal to the tumor should also be recorded. In
large adenocarcinomas, the exact preoperative location of
the tumor may be difficult to determine. One of the
pathologists roles is to elucidate the location of the tumor
and to determine whether it is gastric or esophageal in
origin.
The squamocolumnar junction (Z line) and the GEJ may
not be the same in a given specimen. The columnar mucosa
appears velvety and pink, whereas the squamous mucosa is
glistening, smooth, and gray-to-pearly in color. The Z line is
the intersection of glandular and squamous mucosa. The
GEJ is the junction of the tubular esophagus and the cuff of
the stomach, regardless of the type of mucosa present. In
Barrett esophagus, the columnar-type mucosa is localized
proximal to the Z line and appears as continuous or patchy,
pale pink, and finely granular. There are cases of squamous
dysplasia that cannot be readily grossly observed. The
application of Lugol helps to clearly define the dysplastic
portions because the normal squamous mucosa will pick up
the dye; however, the dysplasia or SCC in situ will not.17
Determining the site of origin is discussed below in the
sampling section.
The adventitia appears as irregular soft tissue whose
thickness varies depending on the location and the surgical
technique. The most outer layer, corresponding to the deep
circumferential margin marked with ink, may harbor lymph
nodes. In situations where lymph nodes are not easily
dissected, the adventitia can be stripped and placed in
alcohol or Bouin fixative. After a few hours, the lymph nodes
Arch Pathol Lab MedVol 139, November 2015 Examination of Surgical Specimens of the EsophagusBejarano & Berho 1449
will appear as white areas contrasting with the background
of connective tissue or fat. There is no established minimum
of lymph nodes to be examined in esophagectomy
specimens. Differences in the number of lymph nodes
may be due to the diligence of the laboratory personnel, but
may also be dependent on the type of esophagectomy
technique used by the surgeon. Additionally, the technique
may have consequences on the thickness of the tissue
deeper to the tumor that is attached to the esophagus.
Independent of the surgical technique used, all lymph nodes
found should be completely embedded for microscopic
examination.
Surgical Options
There are 3 types of open esophagectomy approaches,
including the transhiatal esophagectomy (THE), the Ivor
Lewis transthoracic esophagectomy (TTE), and the
McKeown procedure, which is a modified TTE using the
3-field approach. For the THE, a laparotomy is performed to
mobilize the stomach and to carry out a lymphadenectomy
of the left gastric and the celiac nodes. The thoracic
esophagus is obtained by blunt dissection. The TTE
technique also involves a laparotomy with the aforemen-
tioned lymphadenectomy, but a thoracotomy is performed
to remove the esophagus by meticulous dissection around
the esophagus and removal of mediastinal lymph nodes.
From the pathology point of view, the TTE and 3-field
techniques have the advantage of providing more lymph
nodes and a wider radial margin than the THE.18,19
Veeramachaneni et al19 found more lymph nodes in 64
TTE patients than 59 THE patients (13 versus 9; P , .001) in
a study based on operative notes and pathology reports. It
has also been observed that patients undergoing THE were
more likely to require endoscopic dilatation within 6 months
of surgery.20 These findings help explain why most thoracic
surgeons favor TTE or the open 3-field (McKeown)
procedure. This procedure entails dissection initially in the
chest, then the abdomen, with final anastomosis in the
neck. However, no survival benefit has been evident for one
procedure over the other.2022
Today, more and more surgeons are performing
minimally invasive esophagectomies. Both TTE and the
3-field technique can be performed using minimally
invasive techniques. The abdominal portion is performed
by laparoscopy or hand-assisted technique, and the
thoracic portion includes thoracoscopy.22 More recently,
robotic esophagectomy has been used in some centers.
The advantage of minimally invasive esophagectomies
over open procedures is lower morbidity and mortality. In
experienced hands there is no significant difference in the
number and location of harvested lymph nodes, the
ability to achieve R0 resection (absence of residual tumor
at the margin), the ability to establish accurate staging, or
problems with dissemination of tumor.2325 However, it
appears that there are no significant differences in
survival between the open procedures with extended en
bloc lymphadenectomy and minimally invasive esopha-
gectomies techniques.19
Sampling
The tumor should be adequately sampled in order to
ensure that all of the prognostic parameters are determined
at the time of the microscopic examination. Full-thickness
sections of the tumor that include the inked radial
circumferential margin will provide information about the
Figure 3. Adenocarcinoma and its relationship to the gastroesophageal junction (GEJ). A, External surface in which arrows indicate the invagination
between the tubular esophagus and the bag of the stomach, which is also where the peritoneal reflection at the junction of the esophagus and greater
curvature is found, helps to determine the GEJ. B, Adenocarcinoma in a background of Barrett esophagus. The mid portion of the tumor coincides
with the invagination (arrow) that separates the tubular esophagus and the stomach, and thus this is a tumor from the GEJ.
Figure 4. Complete pathologic response (ypT0). A, Area of squamous mucosa with ulceration and inflamed granulation tissue where a carcinoma
existed prior to chemoradiation therapy. B, Cytokeratin stain highlights positive cells and debris at the base of the ulcer that should not be considered
positive for residual carcinoma (hematoxylin-eosin, original magnification 350 [A]; pankeratin cocktail immunostain, original magnification 3100
[B]).

depth of invasion and the status of the margin. Sections of
the tumor and its interface with the nontumoral mucosa
may provide information on the presence of background
dysplasia or Barrett esophagus from which the carcinoma
may have arisen. Similar findings may be encountered by
sampling the squamocolumnar junction and the GEJ. The
presence of a tumor in the GEJ raises the question of
whether it arose in the esophagus from a Barrett back-
ground or if its origin is that of gastric cardia that has
extended proximally into the esophagus. Because the
separation between squamous mucosa and columnar-type
mucosa may not be accurate, the anatomic GEJ must be
sampled. The GEJ can be appreciated as an invagination
between the tubal esophagus and the bag of the stomach,
which is also where the peritoneal reflection at the junction
of the esophagus and greater curvature is found26 (Figure 3).
The College of American Pathologists cancer protocol for
esophagus states the importance of determining the
midpoint of the tumor as a parameter to establish the
origin in the esophagus, stomach, or GEJ.27 In most cases,
however, it is not possible for the pathologist to determine
the exact tumor origin from resection specimens because the
1450 Arch Pathol Lab MedVol 139, November 2015
anatomic divisions of the esophagus are defined by
anatomic boundaries and relationships to other struc-
tures.27,28 Nonetheless, for purposes of staging, tumors
involving the GEJ are classified as esophageal carcinomas,28
and tumors whose midpoints are in the cardia and cross the
GEJ are classified as GEJ tumors.29 Sections away from the
tumor may disclose the presence of lymphovascular
invasion.
In the absence of obvious tumor after a known history of
chemotherapy and/or radiation, areas of scarring should be
evaluated and correlated with the patients diagnostic
endoscopy for the location of the original tumor. Sometimes
the area of interest has been previously tattooed; therefore,
inked areas marked by the endoscopist should be located.
This prevents one from engaging in random aimless
sampling of the esophageal mucosa. Once the area of
interest is identified, it should be sampled entirely to
evaluate for the presence of residual carcinoma.
If intraoperative frozen section examination of the
proximal and distal margins was not obtained, they are to
be sampled after fixation of the esophagus in formaldehyde
Examination of Surgical Specimens of the EsophagusBejarano & Berho
in the same fashion described above, as if they had been
submitted for frozen section.
Sampling of lymph nodes is of utmost importance
because the information they yield is an independent
prognostic factor in the patients outcome.30 Labeling the
lymph nodes according to the region where they were
obtained in the proximal paraesophageal, distal paraesoph-
ageal, perigastric lesser curvature, or perigastric greater
curvature can be done; however, there is no evidence that
this practice influences the prognosis in the case of positive
nodes.31 Although regional lymph nodes extend from
periesophageal cervical nodes to celiac nodes, the current
American Joint Committee on Cancer staging system does
not take into consideration the location of the nodes.
Currently, there are no standards as to the adequacy and/or
the extent of surgical lymphadenectomy in the American
Joint Committee on Cancer staging system.
The proximal and distal margins can be obtained en face
or in a perpendicular fashion, depending on how close the
tumor is to the margin, in order to clearly demonstrate
whether it is free or involved by tumor microscopically.
Inking the closest margin adds accuracy to the evaluation,
and the distance of the tumor from the luminal margins
should be measured.
Histology
Examination of the tumor confirms the histologic type of
carcinoma diagnosed on the preoperative biopsy tissue and
may allow for more accurate classification. Secondly, it
provides the degree of differentiation. For SCCs, the degree
of differentiation is based in part on the ability of tumor cells
to produce keratin. Thus, tumors with abundant production
of keratin tend to be well differentiated, whereas those with
no keratin production may require immunostains such as
p40 to determine their squamous nature and will likely be
poorly differentiated. Using p40 antibody will help in
determining whether a morphologically undifferentiated
tumor is in reality a poorly differentiated SCC. Staining for
p40 is more desirable than using p63 or cytokeratin 5/6
because of the higher specificity of the former.32 The degree
of differentiation of adenocarcinomas is dictated by how
tumor cells arrange forming glands. Tumors that have
greater than 95% of gland formation are well differentiated,
those with 50% to 95% are moderately differentiated, and
those that are solid with 49% or less of gland formation are
classified as poorly differentiated. Because the last category
may lack tubular or glandlike appearance, showing only
solid sheets of tumor, the use of mucicarmine or CDX2
(caudal type homeobox 2) stains will assist in the diagnosis
of adenocarcinoma.
Depth of Invasion
Depth of invasion stages the carcinoma and predicts
lymph node involvement,33 hence the importance of
accurate diagnosis. As discussed for EMR, the muscularis
mucosa varies in thickness and may be duplicated rather
than form a single layer of smooth muscle fibers. It is
important to be aware of these normal variants of the
muscularis mucosa so as not to confuse it with muscularis
propria.9 Stage pT1 is carcinoma limited to the lamina
propria or that which invades up to the submucosa. In pT2,
the tumor reaches the muscularis propria. The esophagus
lacks serosa, with the most external aspect being layers of
connective tissue representing adventitia and whose
involvement is considered pT3. Extension to adjacent
Arch Pathol Lab MedVol 139, November 2015 Examination of Surgical Specimens of the EsophagusBejarano & Berho 1451
structures is pT4. The presence or absence of lymphovas-
cular and perineural invasion should be documented.
Likewise, it is important to diagnose precursor lesions,
such as squamous or glandular dysplasia of the overlying
epithelium.
Margins
Assessment of the margins is one of the main concerns,
because the goal of the procedure is tumor-free margins.
This evaluation is particularly important in resected speci-
mens after chemoradiation, because the presence or absence
of residual tumor at the margin (R0 resection) dictates
recurrence and survival rates.34 R0 corresponds to the
absence of residual tumor, R1 is microscopic residual tumor,
and R2 is macroscopic residual tumor. It appears that a
distance of 5 to 6 cm for proximal and distal margins to the
tumor on both sides is optimal to determine an R0
resection.35,36 The circumferential resection margin for pT3
tumors is more subject to debate as to what is considered
positive. In the United Kingdom, the Royal College of
Pathologists defines a positive radial margin when tumor
cells are present within 1 mm of the margin, whereas the
College of American Pathologists in the United States
considers a positive margin to be defined by only the
presence of tumor cells in contact with the most outer
(usually inked) margin. In a study from the Netherlands, it
was found that the median survival for adenocarcinoma
patients was significantly different when positive and
negative margins were evaluated based on the College of
American Pathologists definition, whereas no difference
was found when the Royal College of Pathologists criteria
were used.18 Similar findings were observed for SCC
patients, in whom there were equal rates of locoregional
recurrence in patients with tumors less than 1 mm and 0
mm from the margin.37 This supports the view that a true
positive margin is defined by the presence of tumor cells at
the most external radial edge of the specimen. In addition,
there is support for the view that there is no difference in
circumferential resection margin involvement when com-
paring types of surgery performed (either THE or TTE),
applying either the College of American Pathologists or the
Royal College of Pathologists criteria.
It is also important to be aware that reporting pT4b status
is now a required element.27,28 The pT4b status indicates that
there is invasion of adjacent structures, such as the aorta,
vertebral body, or trachea.
Lymph Nodes
The pathologic staging of lymph nodes is a consistent
prognostic parameter. It is based on the number of lymph
nodes independent of the location. Therefore, the more
lymph nodes sampled, the more accurate the prediction of
survival.30 The pathologist must indicate the presence of
extracapsular extension in positive lymph nodes because it
identifies patients with poor long-term prognosis.38 Con-
troversial theories regarding the significance of lymph node
ratios (dividing the number of positive lymph nodes by the
total number of dissected nodes)39 and their size40 have been
proposed as better predictors of patient outcome than the
number of lymph nodes alone. However, this has yet to be
validated.28
Tumor Regression
Patients with esophageal carcinoma may benefit from
chemoradiation before esophagectomy. The goal of neo-

Figure 5. Mucin after therapy. Pools of mucin in the absence of
carcinoma cells should not be considered as residual adenocarcinoma
(hematoxylin-eosin, original magnification 3100).
Figure 6. Effects of irradiation. A, Single irradiated stromal cell with
elongated and atypical hyperchromatic nucleus. The presence of
abundant cytoplasm helps to distinguish it from a residual carcinoma
cell. B, Group of epithelial cells in an angulated (infiltrative) pattern
showing marked nuclear atypia and hyperchromasia with prominent
nucleoli and high nucleus to cytoplasm ratio, suggesting malignancy
(hematoxylin-eosin, original magnification 3400).
1452 Arch Pathol Lab MedVol 139, November 2015
adjuvant therapy is to downstage the tumor and assess
tumor biology, thus increasing the possibility of complete
resection; assess for any development of metastases; and
decrease rates of recurrence.41 Complete histologic response
reaches 30% for both SCC42 and adenocarcinomas.4345 It is
critical for pathologists to review the treated specimens very
carefully. Examination of these specimens can be particu-
larly challenging because changes in the tissues and tumors
may create confusion. The benign mucosa may show
spongiosis, acantholysis, degeneration, and reactive atypia
that mimic carcinoma or dysplasia. The stroma cells also
may appear enlarged, showing bizarre and irregular nuclei
and multinucleation accompanying elastosis and marked
inflammation. Blood vessels tend to show intimal prolifer-
ation, nucleomegaly, thrombosis, and telangiectasis. Judi-
cious immunohistochemical staining for keratin would be
negative in the mesenchymal atypical cells, arguing against
the presence of carcinoma. The lymph nodes may become
atrophic, and thus the number of lymph nodes retrieved can
be low. Moreover, they may show fibrosis and depletion of
the lymphoid population.43
An additional difficult task is determining whether
residual tumor is indeed still present. If present, the tumor
cells nuclei may appear very irregular, with clumping of
chromatin or bizarre forms showing vacuoles and popcorn-
like appearance with multilobation. The architecture is also
distorted with the presence of irregular tubules and
individual cells in a fibrotic stroma. The changes are so
marked that a carcinoma originally classified as well
differentiated may now be erroneously labeled as poorly
differentiated.
In cases in which there are no viable tumor cells, the
presence of necrosis or pools of mucin44 should not be
regarded as residual carcinoma, but rather as a sign of
complete response (ypT0). Keratin flakes may be observed
in relation to an ulcer where the tumor was. Although they
may show positivity for keratin immunostain, these are not
indicative of residual tumor (Figure 4). Likewise, lymph
nodes showing necrosis, fibrosis, or pools of acellular mucin
are not to be considered positive for metastasis (ypN0;
Figure 5). Distinguishing reactive stromal cells from residual
carcinoma cells after chemoradiation may be difficult.
Although atypical and enlarged, stromal cells usually show
abundant cytoplasm, whereas irradiated carcinoma cells
may show an infiltrative pattern, marked nuclear atypia, and
high nucleus to cytoplasm ratio. Therefore, examination
with a medium- to high-power objective is often required.
Also, residual carcinoma cells are often embedded in deep
tissue and in vascular spaces (Figure 6).
Armed with this information, the pathologist can now
grade the response. There are at least 3 grading systems
based on the subjective amount of residual tumor associated
with the presence or absence of fibrosis.4648 A simple
system applied for colorectal carcinomas49 produces a good
interobserver reproducibility: 0, no viable cancer cells
(complete response); 1, single cells or small groups of
cancer cells (moderate response); 2, residual cancer out-
grown by fibrosis (minimal response); and 3, no tumor kill
extensive residual cancer (poor response).
Biologic Targeted Therapies
For adenocarcinoma, the pathologist will select a tissue
block on which immunohistochemical or in situ hybrid-
ization for Her2/neu will be performed. The rationale for
this analysis is based on the applicability of targeted
Examination of Surgical Specimens of the EsophagusBejarano & Berho
 Immunohistochemistry Scoring for HER2 in Gastric and Gastroesophageal Junction Cancer,
by Type of Diagnostic Specimena
Score Surgical Specimen
0 No reactivity or membranous reactivity in ,10% of
tumor cells
1 Faint or barely perceptible in 10% of tumor cells;
cells are reactive only in part of their membrane
2 Weak to moderate complete, basolateral or lateral
membranous reactivity in 10% of tumor cells
3 Strong complete, basolateral or lateral membranous
reactivity in 10% of tumor cells
a
Reprinted from Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for
treatment of HER2-positive advanced gastric or gastro-esophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.
Lancet. 2010; 376:687697. Copyright (2010) with permission from Elsevier.50
b
Requires fluorescent in situ hybridization or chromogen in situ hybridization for confirmation.
therapy for upper gastrointestinal tract adenocarcinomas
with the use of trastuzumab (Herceptin, Genentech, San
Francisco, California), a monoclonal antibody of proven
therapeutic benefit offering improved overall survival when
used in conjunction with chemotherapy. Unfortunately,
this is not applicable to SCCs. The target is the receptor for
Her2 in the membrane of tumor cells. As in breast
carcinomas, esophageal adenocarcinomas may overexpress
Her2, which is associated with its genetic amplification in
segments of chromosome 17, and is thus a predictive
biomarker for therapy with trastuzumab. This discovery
came to light in an international study published in 2010
known as the ToGA (Trastuzumab for Gastric Cancer) trial,
in which 135 laboratories in 24 countries participated.50
Patients were selected who had stage IV (locally advanced,
inoperable, and/or metastatic) adenocarcinomas of the GEJ
and stomach, and whose tumors showed 3 immunoreac-
tivity for Her2 and/or showed fluorescent in situ hybrid-
ization positivity for Her2/CEN-17 2.0. Tumors of the
GEJ showed 33.2% expression or amplification of Her2,
whereas gastric carcinomas showed 20.9% (P , .001). For
both organs, the expression was much more frequently in
the intestinal than in the diffuse or mixed types. In some
centers, chromogenic in situ hybridization is also per-
formed, providing results similar to those of fluorescent in
situ hybridization. Assessment of immunohistochemical
positivity in esophageal adenocarcinomas is different than
that of breast carcinomas, depending on whether the
material analyzed is biopsy tissue or a resected tumor
(Table).
Pathologists have to keep in mind that staining of the
cytoplasm and the basal aspect of the cell alone should not
be considered positive, and that any component of intestinal
metaplasia should be considered in the evaluation. In
addition, heterogeneity within the same tumor and between
the primary carcinoma and its metastases is not infrequent.
Other than Her2, there is no other biologic esophageal
marker currently used in clinical practice.
Finally, providing tissue for research in a standardized
manner is very important to facilitate the investigative
efforts leading to the future discovery of predictors of tumor
response to neoadjuvant therapy and other genetic finger-
prints.51 However, allocation of samples for tissue banking
research is contingent upon the amount of tissue needed for
Arch Pathol Lab MedVol 139, November 2015
Biopsy Specimen Assessment
No reactivity or no membranous reactivity in any Negative
tumor cell
Tumor cell cluster with a faint or barely perceptible Negative
membranous reactivity regardless of percentage
of tumor cells stained
Tumor cell cluster with a weak to moderate Equivocalb
complete, basolateral or lateral membranous
reactivity regardless of percentage of tumor cells
stained
Tumor cell cluster with a strong complete, Positive
basolateral or lateral membranous reactivity
regardless of percentage of tumor cells stained
diagnosis, as well as the evaluation of the parameters
reviewed in this article.
CONCLUSION
The role of the pathologists examination of esophageal
surgical specimens is more important than ever. Knowl-
edge of the variants in the microanatomy of the esophagus
will prevent diagnostic pitfalls. The pathologist must also
become more proficient in evaluating specimens from
more conservative procedures, such as EMRs, in order to
provide the gastroenterologist with the information need-
ed to determine the appropriate care for the patient.
Pathologists use immunohistochemistry to determine the
histologic nature of a tumor in the esophagus and provide
information regarding biomarker expression for therapeu-
tic purposes. The role of the pathologist is pivotal in
assessing diagnostic parameters and staging criteria in
surgically obtained esophageal specimens. This informa-
tion is crucial because it forms the basis for determining the
patients prognosis and therapeutic decisions in order to
prolong survival.
The authors wish to thank Elektra McDermott for her editorial
assistance.
References
1. Lagergren J. Adenocarcinoma of oesophagus: what exactly is the size of the
problem and who is at risk? Gut. 2005;54(suppl 1):i1i5.
2. Liu L, Hofstetter WL, Rashid A, et al. Significance of the depth of tumor
invasion and lymph node metastasis in superficially invasive (T1) esophageal
adenocarcinoma. Am J Surg Pathol. 2005;29(8):10791085.
3. Estrella JS, Hofstetter WL, Correa AM, et al. Duplicated muscularis
mucosae invasion has similar risk of lymph node metastasis and recurrence-free
survival as intramucosal esophageal adenocarcinoma. Am J Surg Pathol. 2011;
35(7):10451053.
4. Alvarez Herrero L, Pouw RE, van Vilsteren FG, et al. Risk of lymph node
metastasis associated with deeper invasion by early adenocarcinoma of the
esophagus and cardia: study based on endoscopic resection specimens.
Endoscopy. 2010;42(12):10301036.
5. Fitzgerald RC, di Pietro M, Ragunath K, et al. British Society of
Gastroenterology guidelines on the diagnosis and management of Barretts
oesophagus. Gut. 2014;63(1):742.
6. Soetikno RM, Gotoda T, Nakanishi Y, Soehendra N. Endoscopic mucosal
resection. Gastrointest Endosc. 2003;57(4):567579.
7. Montgomery E, Bronner MP, Goldblum JR, et al. Reproducibility of the
diagnosis of dysplasia in Barrett esophagus: a reaffirmation. Hum Pathol. 2001;
32(4):368378.
8. Nagai K, Noguchi T, Hashimoto T, Uchida Y, Shimada T. The organization
of the lamina muscularis mucosae in the human esophagus. Arch Histol Cytol.
2003;66(3):281288.
Examination of Surgical Specimens of the EsophagusBejarano & Berho 1453
 9. Abraham SC, Krasinskas AM, Correa AM, et al. Duplication of the
muscularis mucosae in Barrett esophagus: an underrecognized feature and its
implication for staging of adenocarcinoma. Am J Surg Pathol. 2007;31(11):1719
1725.
10. Coco DP, Goldblum JR, Hornick JL, et al. Interobserver variability in the
diagnosis of crypt dysplasia in Barrett esophagus. Am J Surg Pathol. 2011;35(1):
4554.
11. Ormsby AH, Petras RE, Henricks WH, et al. Observer variation in the
diagnosis of superficial oesophageal adenocarcinoma. Gut. 2002;51(5):671676.
12. Odze RD. Diagnosis and grading of dysplasia in Barretts oesophagus. J
Clin Pathol. 2006;59(10):10291038.
13. Kaye PV, Haider SA, Ilyas M, et al. Barretts dysplasia and the Vienna
classification: reproducibility, prediction of progression and impact of consensus
reporting and p53 immunohistochemistry. Histopathology. 2009;54(6):699712.
14. Lomo LC, Blount PL, Sanchez CA, et al. Crypt dysplasia with surface
maturation. a clinical, pathologic, and molecular study of a Barretts esophagus
cohort. Am J Surg Pathol. 2006;30(4):423435.
15. Mitsuhashi T, Lauwers GY, Ban S, et al. Post-gastric endoscopic mucosal
resection surveillance biopsies: evaluation of mucosal changes and recognition of
potential mimics of residual adenocarcinoma. Am J Surg Pathol. 2006;30(5):650
656.
16. Novis DA, Zarbo RJ. Interinstitutional comparison of frozen section
turnaround time: a College of American Pathologists Q-Probes study of 32868
frozen sections in 700 hospitals. Arch Pathol Lab Med. 1997;121(6):559567.
17. Mori M, Adachi Y, Matsushima T, Matsuda H, Kuwano H, Sugimachi K.
Lugol staining pattern and histology of esophageal lesions. Am J Gastroenterol.
1993;88(5):701705.
18. Verhage RJJ, Zandvoort HJA, ten Kate FJW, van Hillegersberg R. How to
define a positive circumferential resection margin in T3 adenocarcinoma of the
esophagus. Am J Surg Pathol. 2011;35(6):919926.
19. Veeramachaneni NK, Zoole JB, Decker PA, Putnam JB, Meyers BF. Lymph
node analysis in esophageal resection: American College of Surgeons Oncology
Group Z0060 trial. Ann Thorac Surg. 2008;86(2):418421.
20. Chang AC, Ji H, Birkmeyer NJ, Orringer MB, Birkmeyer JD. Outcomes after
transhiatal and transthoracic esophagectomy for cancer. Ann Thorac Surg. 2008;
85(2):424429.
21. Omloo JMT, Lagarde SM, Hulscher JBF, et al. Extended transthoracic
resection compared with limited transhiatal resection for adenocarcinoma of the
mid/distal esophagus: five-year survival of a randomized clinical trial. Ann Surg.
2007;246(6):9921001.
22. Feith M, Stein HJ, Siewert JR. Adenocarcinoma of the esophagogastric
junction: surgical therapy based on 1602 consecutive resected patients. Surg
Oncol Clin N Am. 2006;15(4):751764.
23. Yamamoto M, Weber JM, Karl RC, Meredith KL. Minimally invasive surgery
for esophageal cancer: review of the literature and institutional experience.
Cancer Control. 2013;20(2):130137.
24. Hulscher JB, van Sandick JW, de Boer AG, et al. Extended transthoracic
resection compared with limited transhiatal resection for adenocarcinoma of the
esophagus. N Engl J Med. 2002;347(21):16621669.
25. Smithers BM, Gotley DC, Martin I, Thomas JM. Comparison of the
outcomes between open and minimally invasive esophagectomy. Ann Surg.
2007;245(2):232240.
26. Ibrahim NBN. Guidelines for handling oesophageal biopsies and resection
specimens and their reporting. J Clin Pathol. 2000;53(2):8994.
27. Washington K, Berlin J, Branton P, et al. Protocol for the examination of
specimens from patients with carcinoma of the esophagus. College of American
Pathologists Web site. http://www.cap.org/apps/docs/committees/cancer/cancer_
protocols/2013/Esophagus_13protocol_3112.pdf. Accessed January 22, 2015.
28. Digestive system. In: Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL,
Trotti A, eds. AJCC Cancer Staging Manual. 7th ed. New York, NY: Springer;
2010:129144.
29. Chandrasoma P, Wickramasinghe K, Ma Y, DeMeester T. Adenocarcinomas
of the distal esophagus and gastric cardia are predominantly esophageal
carcinomas. Am J Surg Pathol. 2007;31(4):569575
30. Gu Y, Swisher SG, Ajani JA, et al. The number of lymph nodes with
metastasis predicts survival in patients with esophageal or esophagogastric
junction adenocarcinoma who receive preoperative chemoradiation. Cancer.
2006;106(5):10171025.
1454 Arch Pathol Lab MedVol 139, November 2015
31. Ide H, Nakamura T, Hayashi K, et al. Esophageal squamous cell
carcinoma: pathology and prognosis. World J Surg. 1994;18(3):321330.
32. Tatsumori T, Tsuta K, Masai K, et al. p40 is the best marker for diagnosing
pulmonary squamous cell carcinoma: comparison with p63, cytokeratin 5/6,
desmocollin-3, and sox2. Appl Immunohistochem Mol Morphol. 2014;22(5):
377382.
33. Rice TW, Zuccaro G Jr, Adelstein DJ, Rybicki LA, Blackstone EH,
Goldblum JR. Esophageal carcinoma: depth of tumor invasion is predictive of
regional lymph node status. Ann Thorac Surg. 1998;65(3):787792.
34. Siersema PD and van Hillegerberg R. Treatment of locally advanced
esophageal cancer with surgery and chemoradiation. Curr Opin Gastroenterol.
2008;24(4):535540.
35. DiMusto PD, Orringer MB. Transhiatal esophagectomy for distal and cardia
cancers: implications of a positive gastric margin. Ann Thorac Surg. 2007;83(6):
19931999.
36. Barbour AP, Rizk NP, Gonen M, et al. Adenocarcinoma of the
gastroesophageal junction: influence of esophageal resection margin and
operative approach on outcome. Ann Surg. 2007;246(1):18.
37. Chao YK, Yeh CJ, Chang HK, et al. Impact of circumferential resection
margin distance on locoregional recurrence and survival after chemoradiotherapy
in esophageal squamous cell carcinoma. Ann Surg Oncol. 2011;18(2):529534.
38. Lagarde SM, ten Kate FJ, de Boer DJ, Busch ORC, Obertop H, van Lanschot
JJB. Extracapsular lymph node involvement in node-positive patients with
adenocarcinoma or the distal esophagus or gastroesophageal junction. Am J
Surg Pathol. 2006;30(2):171176.
39. Lagarde SM, Reitsma JB, de Castro SMM, ten Kate FJW, Busch ORC, van
Lanschot JJB. Prognostic nomogram for patients undergoing oesophagectomy for
adenocarcinoma of the oesophagus or gastro-oesophageal junction. Br J Surg.
2007;94(11):13611368.
40. Dhar DK, Hattori S, Tonomoto Y, et al. Appraisal of a revised lymph node
classification system for esophageal squamous cell cancer. Ann Thorac Surg.
2007;83(4):12651272.
41. Gebski V, Burmeister B, Smithers BM, Foo K, Zalcberg J, Simes J. Survival
benefits from neoadjuvant chemoradiotherapy or chemotherapy in oesophageal
carcinoma: a meta-analysis. Lancet Oncol. 2007;8(3):226234.
42. Brucher BLDM, Becker K, Lordick F, et al. The clinical impact of
histopathologic response assessment by residual tumor cell quantification in
esophageal squamous cell carcinomas. Cancer 2006;106(10):21192127.
43. Chang F, Deere H, Mahadeva U, George S. Histopathologic examination
and reporting of esophageal carcinomas following preoperative neoadjuvant
therapy. Am J Clin Pathol. 2008;129(2):252262.
44. Chirieac LR, Swisher SG, Correa AM, et al. Signet-ring cell or mucinous
histology after preoperative chemoradiation and survival in patients with
esophageal or esophagogastric junction adenocarcinoma. Clin Cancer Res.
2005;11(6):22292236.
45. Heath EI, Burtness BA, Heitmiller RF, et al. Phase II evaluation of
preoperative chemoradiation and postoperative adjuvant chemotherapy for
squamous cell and adenocarcinoma of the esophagus. J Clin Oncol. 2000;
18(4):868876.
46. Mandard AM, Dalibard F, Mandard JC, et al. Pathologic assessment of
tumor regression after preoperative chemoradiotherapy of esophageal carcinoma:
clinicopathologic correlations. Cancer. 1994;73(11):26802686.
47. Wu TT, Chirieac LR, Abraham SC, et al. Excellent interobserver agreement
on grading the extent of residual carcinoma after preoperative chemoradiation in
esophageal and esophagogastric junction carcinoma: a reliable predictor for
patient outcome. Am J Surg Pathol. 2007;31(1):5864.
48. Hermann RM, Horstmann O, Haller F, et al. Histomorphological tumor
regression grading of esophageal carcinoma after neoadjuvant radiochemother-
apy: which score to use? Dis Esophagus. 2006;19(5):329334.
49. Ryan R, Gibbons D, Hyland JM, et al. Pathological response following
long-course neoadjuvant chemoradiotherapy for locally advanced rectal cancer.
Histopathology. 2005;47(2):141146.
50. Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combination
with chemotherapy versus chemotherapy alone for treatment of HER2-positive
advanced gastric or gastro-esophageal junction cancer (ToGA): a phase 3, open-
label, randomised controlled trial. Lancet. 2010;376(9742):687697.
51. Bonavina L, Laface L, Picozzi S, et al. Proposal of a punch biopsy protocol
as a pre-requisite for the establishment of a tissue bank from resected esophageal
tumors. Pathol Oncol Res. 2010;16(3):457460.
Examination of Surgical Specimens of the EsophagusBejarano & Berho