Abstract
Accurate pathologic information in an endoscopic resection specimen of early gastric cardiac cancer dictates the patient management plan and should be acquired through a close collaboration between the endoscopist and the pathologist to determine (1) tumor location, size, and relationship with the esophagogastric junction; (2) presence or absence of ulceration; (3) histopathological type; (4) depth of invasion; (5) lymphovascular and perineural invasion; (6) status of horizontal and vertical margins; (7) other gastric diseases, and (8) distal esophageal lesions.
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Introduction
Endoscopic resection of neoplasms in the stomach is a type of minimally invasive procedures and has a number of advantages over open surgical resection in terms of lower cost, shorter duration of hospital stay, less blood loss, fewer complications, better preservation of gastric function, and more rapid recovery after the procedure [1]. Endoscopic resection, such as endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD), has been widely accepted and used for the treatment of gastric epithelial neoplasms, especially for early gastric carcinoma (EGC) with negligible risk of lymph node metastasis [2]. Since ESD is significantly more effective than EMR with respect to en bloc resection, lower local recurrence, and complete histopathological assessment of the specimen, most gastric superficial neoplasms have been resected with ESD techniques primarily in East Asian countries [3]. In our practice, most early gastric cardiac carcinomas are resected endoscopically with ESD. As such, accurate histopathologic evaluation of the ESD specimen is critically important and provides the critical information not only on the accurate diagnosis, tumor stage, and associated diseases but also on the adequacy of the ESD resection, margin status, and the presence or absence of risk factors for nodal metastasis to guide patient further triage and management. Over the past several years, we have performed a considerable number of pathologic evaluations of ESD specimens at the Nanjing Drum Tower Hospital, armed with the latest versions of the practice guidelines and protocols issued by the Chinese Society of Digestive Endoscopy [4], the Japan Gastroenterological Endoscopy Society (JGES), and the Japanese Gastric Cancer Association (JGCA) [2, 5] and the World Health Organization WHO Classification of Tumours of the Digestive System (4th edition) [6]. In this chapter, we will present our experience on the methods and protocols on histopathologic assessment of the ESD specimen on early gastric cardiac carcinoma.
Processing of ESD Specimens
Once an early gastric carcinoma is resected with the ESD technique, it is the responsibility of the endoscopist, not pathologist, to immediately start appropriate tissue processing within the endoscopic suit, where the fresh specimen is slightly stretched out, pinned down on a dental wax plate with accurate orientation, photographed, and then immediately immersed in the 10% neutral buffered formalin solution. The tissue ischemic time elapsed between complete resection and formalin fixation should be as short as possible (shorter than 30 min) to minimize tissue degradation and better preserve DNA and RNA molecules. As a general rule, the immersion duration in formalin should be 12–48 h at room temperature to ensure adequate fixation for possible molecular studies in the future.
Tips for the endoscopist on initial ESD tissue processing:
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1.
Because the muscularis mucosae will contract after endoscopic resection, the most important thing on immediate fresh ESD specimen handling is to preserve the intact normal anatomy by slightly stretching the fresh tissue and pinning it down on the plate. The endoscopist should mark clearly the correct orientation of the specimen with appropriate markings so that the pathologist will evaluate the dissection margins accurately, minimizing the incidence of reporting false-positive or false-negative margins. We use tweezers together with pins to make the stretching process easier (Figs. 12.1 and 12.2).
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2.
Stainless steel thin pins , such as either insect specimen fixation pins (size 1# or 2#) or acupuncture needles, are good choices for pinning down an ESD specimen with minimal tissue distortion or damage.
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3.
The polystyrene board can be easily found and used as an ideal plate for ESD-resected specimen handling (Fig. 12.3).
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Neutral buffered formalin means 10% volume/formalin in water (approx. 4% formaldehyde), pH 7.4. The specimen should be fixed with at least ten times more 10% neutral buffered formalin solution. Therefore, a large container should be used for each ESD specimen.
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5.
During the initial fresh specimen processing procedure in the endoscopic suit, the specimen should be kept wet with normal saline solution to prevent the fresh specimen from drying up and maintain the tissue in vivo physiology before immersing into the formalin solution for a better preservation.
In a well-sealed container, the specimen will be delivered to the pathologist for further processing, such as sectioning of the fully fixed specimen (over 10–24 h), macroscopic photography before and after sectioning, and histology tissue processing.
Once fixed well, the specimen is removed from the plate, and all resection margins are inked, ideally with different colors. Before sectioning, the mucosal surface of an ESD specimen should be washed first to remove mucus, residual food particles, necrotic debris, blood clots, etc. Then, the specimen will be carefully inspected, and the gross characteristics of the tumor and the adjacent uninvolved mucosal tissue should be described, measured, and recorded, including:
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1.
The specimen size
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Mucosal surface color (discoloration of the mucosal surface of the lesion, compared to the surrounding normal mucosa)
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3.
The presence or absence of ulceration (UL, lesions with ulcerations or scarring from the previous or current ulcer)
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4.
The location of the tumor epicenter, size, shape, and color
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The distance (in millimeter) of the tumor edge to peripheral and deep margins of resection
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6.
The relationship and distance (in millimeter) of the tumor edge to the gastroesophageal junction (GEJ)
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7.
Any mucosal lesions in the adjacent gastric cardia and the distal esophagus, especially columnar-lined esophagus
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8.
Whether or not the tumor invades into the distal esophagus, if so, the relationship and distance between the tumor in the cardia and the tumor in the distal esophagus, in association with columnar-lined esophagus and other esophageal lesions
Among these observations, the tumor size, margin, and shape (protruding, flat, or ulceration) and the relationship to the GEJ are particularly important. For example, the size of the neoplasm with or without ulcer is critical for the decision of endoscopic therapy and additional surgery [2]. The distance from the tumor edge to the nearest resected margin may help determine the adequacy of the ESD excision and the need for adjuvant or additional resection therapy.
Macroscopic photography should be ta ken before and after sectioning (Fig. 12.4).
Macroscopic appearances of an early tumor are reclassified, according to the Paris endoscopic classification [7]. Type 0 early tumors are not the same as superficial lesions of the Borrmann classification (proposed for advanced gastric tumors in 1926, which includes Types I–IV) [5]. For Type 0 early tumors , the gross types of the superficial early tumor lesions are divided into polypoid (0-Ip and 0-Is) and non-polypoid subtypes. The non-polypoid subtypes include lesions with a small variation of superficial (0-IIa, slightly elevated; 0-IIb, flat; 0-IIc, depressed) and excavated lesions (0-III). Superficial tumors with O-II or more components sho uld have all components recorded in the order of the surface area occupied, e.g., 0-IIa + IIc (Figs. 12.5 and 12.6). After gross tumor photography, the specimen is cut longitudinally into parallel slices. The first incision is made to allow pathological examination of the lesion with the minimal distance between the margin of the lesion and the lateral edge of a specimen. Then, further sections are made parallel to the first one at interval of 2.0–3.0 mm (Fig. 12.7a–c). As shown in Fig. 12.7a, one can imagine a line tangential to the tumor margin that is the closest to the horizontal margin of the specimen (mucosal dissection margin ) and make the first cut perpendicular to this tangential line.
After specimen sectioning is completed, the tissue slices will be sequentially placed in tissue embedding cassettes for paraffin embedding in the correct order of sequence for histology reassembling of the resected tumor. Paraffin tissue blocks will be sliced at 4 μm in thickness and stained with a routine hematoxylin-eosin (H&E) stain.
Tips:
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1.
Each tissue slice should be rotated by 90° before being placed into an embedding cassette (Fig. 12.8a).
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2.
To ensure the straightness and correct order, we use a thin sponge to push the tissue slice to the side of an embedding cassette (Fig. 12.8b).
To appropriately reconstruct the extent of intramucosal spread and depth of invasion (Fig. 12.9), it is d esirable to take macroscopic photographs of the fixed, sliced specimen before placing tissue slices into cassettes for comparison and tumor mapping purposes.
Histopathological Report
The essential elements in a pathology report on an ESD specimen include:
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The size and number of the specimens
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The macroscopic type of the tumor (according to the Paris classification)
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The size of the tumor (longest and shortest dimensions)
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The histological type of the tumor (based on the WHO classification)
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Depth of tumor invasion
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The presence or absence of lymphovascular invasion
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Perineural invasion
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The involvement of horizontal and vertical margins (HM and VM) by a dysplastic or invasive lesion
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Nonneoplastic pathologic lesions in the adjacent uninvolved tissue
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Any invasion into the distal esophagus
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The presence or abs ence of columnar-lined esophagus and dysplasia in the distal esophagus [4, 5]
According to the WHO diagnostic criteria [6], EGC is defined as an invasive neoplastic epithelial lesion of the stomach confined to the mucosa (M) and/or submucosa (SM), regardless of the lymph node status. Histopathological types of gastric adenocarcinoma include five main categories [tubular, papillary, mucinous, poorly cohesive (including signet ring cell carcinoma and other variants), and mixed carcinomas and rare entities (such as adenosquamous carcinoma, squamous cell carcinoma, hepatoid adenocarcinoma, neuroendocrine carcinoma, carcinoma with lymphoid stroma, etc.)]. For early gastric cardiac carcinoma, most tumors are tubular and/or papillary adenocarcinomas. Because papillary adenocarcinoma is prone to lymph node metastasis and usually with a poor prognosis, the diagnostic criteria should be strictly followed [8]. Unlike distal gastric non-cardiac carcinomas, gastric cardiac carcinoma is more heterogeneous and more likely to be composed of uncommon histological types, such as high-grade neuroendocrine carcinoma, adenosquamous carcinoma, mixed adeno-neuroendocrine carcinoma, hepatoid adenocarcinoma, pancreatic acinar-like adenocarcinoma, carcinoma with lymphoid stroma, choriocarcinoma, squamous cell carcinoma, undifferentiated carcinoma, and carcinosarcoma [8, 9].
Gastric carcinoma categories are similar between WHO and Japanese classifications with only slight differences. According to the Japanese classification of gastric carcinoma, the third English edition [5], all tumors are categorized as either differentiated or undifferentiated (Nakamura’s classification ). Well- or moderately differentiated tubular and papillary adenocarcinomas of the WHO classification are classified as differentiated cancers, whereas signet ring cell and poorly differentiated adenocarcinomas and others are classified as undifferentiated. Furthermore, when multiple histopathological types coexist, each histopathological type should be recorded, in descending order of relative proportions of each type within the same lesion (e.g., tubular > papillary > poorly differentiated, etc.) (Fig. 12.10).
The depth of tumor invasion needs to be measured and reported in micrometer, including the deepest point at which the cancer has infiltrated. When submucosal invasion is present, the tumor submucosal invasion depth should be measured as the distance from the lowest edge of the muscularis mucosae to the deepest point of an invading cancer. The depth of submucosal penetration is classified into two su bgroups: SM1 (<500 μm penetration into submucosa or T1b1) and SM2 (≥500 μm or T1b2) (Fig. 12.11) [2, 5]. The abovementioned vertical infiltration distance is measured using a microscope equipped with an ocular scale bar. If the muscularis mucosae is obscure and indiscernible due to tumor ulceration or an ulcerated scar within the lesion, an imaginary continuous line may need to be mentally drawn between the residual muscularis mucosae in the adjacent mucosa at the two sides of an ulcerated lesion for the measurement of the vertical invasion depth. Immunohistochemical staining with the anti-desmin antibody is also useful in identifying the residual muscularis mucosae (Figs. 12.11 and 12.12). In cases with massiv ely submucosal invasive tumors, the depth of submucosal invasion is measured (estimated) from the surface of the tumor to the deepest invasion point (Fig. 12.13).
When the vertical margin is involved, there is no need for measuring the invasive depth, but the possibility of deeper invasion should be described in the pathological report. In cases in which muscularis mucosae is irregular or partially destroyed by cancer infiltration as part of desmoplasia, it is hard to choose a reliable method to measure the depth of submucosal invasion . In fact, discrepancies in invasion depth measurement may be related to different measurement methods. For instance, Kim et al. [10] recently reported that differences in depth of tumor invasion depended upon whether the measurement was started from the bottom of the muscularis mucosae (which was termed as the classic method in their paper) or from an imaginary line at the muscularis mucosae (as the alternative method). In their study, the mean depth of invasion was greater in the nodal positive group than in the nodal negative group when the depth of invasion was evaluated by the alternative method (P = 0.012). However, there was no significant difference between the groups when the classic measurement method was used (P = 0.128). In addition, they categorized the muscularis mucosae in submucosal invasive EGC as normal with well-preserved muscularis mucosae (Fig. 12.14a), as hypertrophic with thickened or expanded muscularis mucosae secondary to the infiltration of tumor cells (Fig. 12.14b), or as discontinuous with irregular muscularis mucosae due to disruption of its continuity by infiltrating tumor cells (Fig. 12.14c) or even disappeared in the cancerous invasive area (Fig. 12.13). Receiver operating characteristic (ROC) curve analysis demonstrated a difference between the methods of measurement as well. The area under the ROC curve using the alternative method was greater than the classic method (0.652 vs. 0.620) among all morphologic patterns of muscularis mucosae. The differences were magnified when evaluating discontinuous and hypertrophic type (0.672 vs. 0.601), compared to normal muscularis mucosa (0.543 vs. 0.581), separately. Finally, they recommended measuring the submucosal depth of invasion from an imaginary line of the muscularis mucosae in patients with irregular muscularis mucosae (discontinuous, hypertrophic) and from the bottom of muscularis mucosae in patients with normal muscularis mucosae.
Ulceration in the stomach is defined as a loss of mucosal tissue to the muscularis mucosae and, sometimes, deep to the submucosa and even muscularis propria. Oftentimes, it is necessary to determine whether or not tumor ulceration or an ulcerated scar is present within the lesion for accurate evaluation on whether or not an ESD resection has been curative. In this setting, the diagnosis of intralesional ulceration becomes critically important. Ulcer should not be confused and misdiagnosed with erosion that does not involve the muscularis mucosae.
It must be pointed out that discrepancies in tumor size and other risk factors related to lymph node metastasis are considerable between pre- and post-ESD procedures for EGC. A recent multicenter Korean study of 737 EGC cases reported an overall discrepancy rate of 20.1% (148/737) [10], most frequently related to the tumor size (8.7%, 64/736), depth of deep submucosal invasion (SM2, 6.9%, 51/737), and ulceration (4.6%, 34/737). The most problematic issue was related to ulceration, in which depth of tumor invasion was significantly deeper in the cases with ulceration than those without. Because depth of invasion was significantly correlated with tumor size, ulceration, differentiation, lymphovascular invasion, and endoscopic tumor gross types, it is critically important for the responsible pathologist to carefully measure and accurately determine the depth of tumor invasion in ESD-resected EGC specimens [10]. Because of 20–30% shrinkage of gastric mucosal tissue after formalin fixation, the most accurate determination of tumor size is best carried out by the ESD endoscopist in the endoscopy unit immediately after the specimen is appropriately pinned down on a plate. The endoscopic differentiation between “ulceration” and “erosion ” is oftentimes difficult, and the histologic diagnosis of ulceration or erosion remains the gold standard.
Assessment of lymphovascular invasion should be carried out using immunohistochemical staining with anti-vascular endothelial antibodies, such as CD31 to identify vessels (v), and anti-lymphatic endothelial antibody (D2–40) for lymphatic channels (ly) (Fig. 12.15). Elastic fiber staining (Elastica van Gieson) is useful for illustration of the elastic fiber in a vessel and identifying intrav ascular cancerous emboli (Fig. 12.16).
Other pathologic changes should also be recorded, such as precancerous lesions (e.g., intraepithelial neoplasia), H. pylori infection, chronic atrophic gastritis, intestinal metaplasia, pancreatic metaplasia, etc. Gastritis cystica profunda involving submucosa should be reported, distinguished with invasive well-differentiated adenocarcinoma, and also thoroughly inspected for the presence or absence of epithelial dysplasia [11]. Any pathologic lesions in the distal esophagus such as columnar-lined esophagus, intestinal metaplasia, dysplasia, and carcinoma unrelated to early gastric cardiac carcinoma must be reported. In our practice, Barrett’s esophagus and distal esophageal Barrett’s adenocarcinoma should be clearly scrutinized in the ESD specimen for the extent of the disease along with the endoscopic/clinical findings. In general, columnar-lined esophagus with intestinal metaplasia within 1 cm above the EGJ line should not be interpreted as Barrett’s esophagus, if given non-specific clinical presentation and unremarkable endoscopic findings. For adenocarcinoma in the distal esophagus, a careful investigation into the origin of this tumor should be meticulously conducted, including identification of the tumor epicenter, the distance and relationship to the EGJ line, associated components of uncommon histology subtypes, and intestinal metaplasia, dysplasia in the adjacent esophageal columnar-lined esophagus. By definition, Barrett’s adenocarcinoma derives from Barrett’s intestinal metaplasia and shows primarily tubular and papillary adenocarcinoma phenotypes but most unlikely presents as adenosquamous carcinoma, signet ring cell carcinoma, mucinous carcinoma, neuroendocrine carcinoma, and carcinoma with lymphoid stroma, among other uncommon types of malignancy, which belongs to gastric cardiac carcinoma [12, 13].
Summary
Optimal pathologic evaluation of the ESD specimen for early gastric cardiac carcinoma is crucial for determination of whether or not additional surgery with lymph node dissection is indicated and how the patient will be triaged and managed. A comprehensive accurate pathology report on the ESD specimen provides valuable information for clinical decision-making and determines (1) the histopathological type, (2) tumor size, (3) whether ulceration is present, (4) depth of invasion, (5) lymphovascular and perineural invasion, (6) horizontal and vertical margin involvement, (7) associated other gastric diseases, and (8) distal esophageal lesions. It requires close communication and collaboration between the endoscopist and the pathologist with the standardized pathologic tissue processing protocol to ensure the best management of patients with early gastric cardiac carcinoma.
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Sun, Q., Huang, Q. (2018). Pathologic Evaluation of Endoscopic Resection Specimens. In: Huang, Q. (eds) Gastric Cardiac Cancer. Springer, Cham. https://doi.org/10.1007/978-3-319-79114-2_12
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