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Examination of Surgical Specimens of the Esophagus.

Esophageal 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 pathologist's 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 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 o'clock. The base 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, submucosa, 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 measurement 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 characterized 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, measuring 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 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 submucosa, 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 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 pathologist's role is expanded to determine the amount of residual tumor burden or tumor regression. Occasionally, 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 pathologist's initial evaluation of the esophagectomy specimen occurs while the patient is still under anesthesia. The decision to reconstruct and anastomose 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 minutes. (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 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 information 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 pathologist's 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 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 aforementioned 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 al (19) 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. (20-22)

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. (23-25) However, it appears that there are no significant differences in survival between the open procedures with extended en bloc lymphadenectomy and minimally invasive esophagectomies 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 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 background 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 found (26) (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 anatomic divisions of the esophagus are defined by anatomic boundaries and relationships to other structures. (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 patient's 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 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 patient's outcome. (30) Labeling the lymph nodes according to the region where they were obtained in the proximal paraesophageal, distal paraesophageal, 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 structures is pT4. The presence or absence of lymphovascular 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 specimens 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 comparing 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) Controversial 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 X100). 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. (43-45) It is critical for pathologists to review the treated specimens very carefully. Examination of these specimens can be particularly 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 proliferation, nucleomegaly, thrombosis, and telangiectasis. Judicious 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-ike 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 mucin (44) 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. (46-48) A simple system applied for colorectal carcinomas (49) 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 outgrown 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 hybridization for Her2/neu will be performed. The rationale for this analysis is based on the applicability of targeted 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+ immunoreactivity for Her2 and/or showed fluorescent in situ hybridization positivity for Her2/CEN-17 [greater than or equal to] 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 performed, 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 fingerprints. (51) However, allocation of samples for tissue banking research is contingent upon the amount of tissue needed for diagnosis, as well as the evaluation of the parameters reviewed in this article.

CONCLUSION

The role of the pathologist's examination of esophageal surgical specimens is more important than ever. Knowledge 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 needed 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 therapeutic purposes. The role of the pathologist is pivotal in assessing diagnostic parameters and staging criteria in surgically obtained esophageal specimens. This information is crucial because it forms the basis for determining the patient's prognosis and therapeutic decisions in order to prolong survival.

The authors wish to thank Elektra McDermott for her editorial assistance.

Please Note: Illustration(s) are not available due to copyright restrictions.

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Pablo A. Bejarano, MD; Mariana Berho, MD

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 (email: bejarap@ccf.org).

Caption: 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 X100).

Caption: 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 X100).

Caption: 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.

Caption: 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 X50 [A]; pankeratin cocktail immunostain, original magnification X100 [B]).

Caption: Figure 5. Mucin after therapy. Pools of mucin in the absence of carcinoma cells should not be considered as residual adenocarcinoma (hematoxylin-eosin, original magnification3100).

Caption: 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 X400).
Immunohistochemistry Scoring for HER2 in Gastric and Gastroesophageal
Junction Cancer, by Type of Diagnostic Specimen (a)

Score        Surgical Specimen             Biopsy Specimen

0        No reactivity or             No reactivity or no
           membranous reactivity in     membranous reactivity in
           <10% of tumor cells          any tumor cell

1+       Faint or barely              Tumor cell cluster with a
           perceptible in [greater      faint or barely
           than or equal to] 10% of     perceptible membranous
           tumor cells; cells are       reactivity regardless of
           reactive only in part of     percentage of tumor cells
           their membrane               stained

2+       Weak to moderate             Tumor cell cluster with a
           complete, basolateral or     weak to moderate
           lateral membranous           complete, basolateral or
           reactivity in [greater       lateral membranous
           than or equal to] 10% of     reactivity regardless of
           tumor cells                  percentage of tumor cells
                                        stained

3+       Strong complete,             Tumor cell cluster with a
           basolateral or lateral       strong complete,
           membranous reactivity in     basolateral or lateral
           [greater than or equal       membranous reactivity
           to] 10% of tumor cells       regardless of percentage
                                        of tumor cells stained

Score     Assessment

0          Negative

1+         Negative

2+       Equivocal (b)

3+         Positive

(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;376:687-697. Copyright
(2010) with permission from Elsevier. (50)

(b) Requires fluorescent in situ hybridization or chromogen in situ
hybridization for confirmation.
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Author:Bejarano, Pablo A.; Berho, Mariana
Publication:Archives of Pathology & Laboratory Medicine
Date:Nov 1, 2015
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