| Abstract|| |
Background: This study aimed to compare CD31, smooth muscle myosin (SMM), and transgelin antibodies for their efficiency in detecting venous invasion (VI) and the nature of free tumor deposits (TDs) in gastric, pancreatic, and colorectal adenocarcinomas. Materials and Methods: Eleven Whipple, 5 gastrectomy, and 3 colectomy specimens and 1 low anterior resection specimen were reviewed and examined, revealing 254 probable foci. Foci were reviewed and divided into 3 types: Type A, the “orphan artery” pattern; Type F, free TDs in the periorgan adipose and connective tissue without an unaccompanied artery; and Type X, a focus that could be detected only with the immunohistochemical procedures mentioned. Results: No foci were positive for CD31. Transgelin staining was more sensitive than SMM staining in all focus types, Type A only and Type F only (P < 0.001, P = 0.001, and P = 0.10, respectively). In free TDs (Type F), 35.7% of the samples were negative for all four stains, and 64.2% of the samples were positive for SMM and transgelin. We did not make the distinction between a metastatic lymph node and VI in positive foci. Conclusion: We conclude that hematoxylin and eosin (H and E) staining is inadequate and that smooth muscle markers, such as transgelin and/or SMM, are more effective than endothelial markers, such as CD31, in revealing VI and lymph node/large extramural invasion.
Keywords: CD31, SMM, transgelin
|How to cite this article:|
Ogut B, Ekinci O, Celik B, Unal ER, Dursun A. Comparison of the efficiency of transgelin, smooth muscle myosin, and CD31 antibodies for the assessment of vascular tumor invasion and free tumor deposits in gastric, pancreatic, and colorectal adenocarcinomas. Indian J Pathol Microbiol 2020;63:25-31
|How to cite this URL:|
Ogut B, Ekinci O, Celik B, Unal ER, Dursun A. Comparison of the efficiency of transgelin, smooth muscle myosin, and CD31 antibodies for the assessment of vascular tumor invasion and free tumor deposits in gastric, pancreatic, and colorectal adenocarcinomas. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Feb 23];63:25-31. Available from: http://www.ijpmonline.org/text.asp?2020/63/1/25/277364
| Introduction|| |
Venous invasion (VI) is a well-defined independent prognostic factor for all cancers, but detection of a vessel that infiltrates into a tumor can be difficult using hematoxylin and eosin (H and E) staining. Histochemical (elastin) methods to visualize the venous wall and immunohistochemical methods (CD31, CD34, and D2-40) to detect endothelium have been developed to facilitate diagnosis and improve accuracy.
In standard H and E sections, there are some well-defined histological characteristics to consider for VI. One characteristic is an orphan arteriole, defined as a well-defined tumor nodule with or without a residual venous wall adjacent to a medium diameter artery. The other characteristic is a protruding tongue, defined as a well-limited protrusion from the tumor to the pericolic fat tissue. Current protocols of the College of American Pathologists suggest using elastin staining when these findings are observed.
Two types of VI are identified in colorectal carcinoma (CRC): (i) extramural (beyond muscularis propria) and (ii) intramural (submucosa or muscularis propria). Extramural VI (EMVI) is an independent adverse prognostic factor, while the significance of intramural VI (IMVI) requires further investigation. In gastric adenocarcinoma, there is no distinction between EMVI and IMVI in the literature.
In CRC, Stage III and Stage II patients with high-risk factors undergo systemic adjuvant therapy. In Stage II patients, VI is one of the factors considered when making the decision for chemotherapy. Therefore, information on VI should be indispensable for pathology reports.
Free tumor deposits (TDs) on perirectal or mesocolic fat tissue were first considered in the TNM stage classification in 1997. The next TNM stage edition chose to evaluate irregular TDs as VI and regular TDs as positive lymph nodes. Additional studies have indicated that TDs are also important prognostic markers., In 2007, cases with nonlymph node metastases but TDs were included in the “pN1c” category, and this classification continues to be used in the current TNM classification.
The aim of this study was to investigate the efficiency of smooth muscle markers (SMM and transgelin) and an endothelial marker (CD31) in IMVI and EMVI in pancreatic, gastric, and colorectal adenocarcinomas. In addition, we investigated the relationship between VI and TD. The results suggest that using smooth muscle markers instead of endothelial markers increases the accuracy of diagnosis, facilitating the recognition of VI in all organ carcinomas.
| Materials and Methods|| |
In this study, 11 pancreaticoduodenectomy, 5 total gastrectomy, and 3 segmental colectomy specimens and 1 low anterior rectum resection specimen diagnosed from 2007 to 2015 at our pathology department were included. For each case, H and E-stained slides were observed by light microscopy to identify any of the suspicious focus types for VI. In total, 254 foci in 79 tissue blocks were selected.
Information such as age, gender, tumor location, tumor diameter, and TNM stage for 20 patients was obtained from our hospital information system.
Resection materials from the selected patients were retrospectively examined for an “orphan arteriole” or “protruding tongue,” and appropriate tumor blocks were determined. In H and E slides, suspected foci were marked with a permanent pen and named like “A1 or F3.” There were not two F3s in one slide. All of the foci had a name, like 173 (case number)-T3 (block code)-A2 (focus name). After this procedure, CD31, transgelin, and SMM markers were studied immunohistochemically in these tumor blocks. On the immunohistochemistry slides, the same foci were marked with the permanent pen by placing the immunohistochemistry slide on the H and E slide and marking all of the foci were selected randomly based on suspected VI. Moreover, the histological type, grade, invasion depth, and stage of the tumor were determined according to the American Joint Committee on Cancer/Union for International Cancer Control 8th edition. EMVI or IMVI categorization of foci and desmoplasia with transgelin was noted. We excluded foci that disappeared in immunohistochemical sections.
We used the same method used in our previous report and added Type F (the latter was based on our findings reported in the manuscript on free TDs, which is in the publication process). Foci were separated into three groups as follows: Type A, the “orphan (ed)” artery pattern, a tumoral focus with a nearby artery unaccompanied by a vein; Type F, free TDs in the periorgan extramural adipose or connective tissue without an unaccompanied artery and evidence of residual lymph node parenchyma (presence of peripheral lymphocyte rim and follicles); and Type X, a focus that only appears after auxiliary immunohistochemical procedures, i.e., foci discovered “collaterally” (without any prediction or suspicion on the H and E slides) in the selected blocks containing foci of the other two types.
Type A and Type F foci have been diagnosed in H and E-stained slides. However, Type X foci could be identified with immunohistochemistry.
CD31 (clone: JC70, ready to use, Ventana Medical Systems Inc.), smooth muscle myosin (clone: SMMS-1, ready to use, Ventana Medical Systems Inc.), and transgelin (clone: 2A10C2, 1:100, Cell Marque) staining was performed on deparaffinized, rehydrated tissue sections obtained from formalin-fixed and paraffin-embedded tissue blocks, using an automated slide stainer (BenchMark XT and BenchMark GX; Ventana Medical Systems Inc., Tucson, AZ, USA). Paraffin-embedded blocks were sectioned, deparaffinized, rehydrated, and blocked with methanolic 3% hydrogen peroxide. Antigen retrieval was performed in ethylenediaminetetraacetic acid buffer. Normal colon wall was used as a positive control for all the markers. Uninterrupted or interrupted circumferential staining around a tumor focus was considered as positive staining based on our previous report.
Immunohistochemical scoring was performed using the following parameters: 3+, positivity in more than 66% of the circumference; 2+, positivity in 33%–66% of the circumference; 1+, positivity in <33% of the circumference; and 0, no staining.
For each focus and each stain, both positive/negative results and the immunohistochemical score (IS) were provided.
For this study, ethics committee approval numbered 17 was obtained from Gazi University Faculty of Medicine Research Ethics Committee on October 12, 2015.
Statistical analysis was performed with SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). Categorical variables were presented as frequencies and percentages and were compared using the Chi-square test and posthoc Chi-square test. Two-tailed values at P < 0.05 were considered statistically significant. Results were considered significant at the P < 0.05 level.
| Results|| |
The patients' clinical data are shown in [Table 1].
Seventy-nine blocks belonging to 20 patients (11 pancreatico duodenectomy, 5 total gastrectomy, and 3 colectomy specimens and 1 low anterior resection specimen) were included in this study. The specimens originated from 14 male (70%) and 6 female (30%) patients aged between 42 and 84 years (mean: 65.7). Tumor size ranged from 0.3 to 7.5 cm (mean, 4.2 cm). The tumors were located in the pancreas (n = 8), duodenum (n = 2), colon (n = 3), rectum (n = 1), corpus of stomach (n = 2), antrum of stomach (n = 1), common bile duct (n = 1), and gastroesophageal junction (n = 2).
According to the WHO criteria published in 2010, the cases were diagnosed as follows: 4 cases as well-differentiated adenocarcinoma, 13 cases as moderately differentiated adenocarcinoma, and 3 cases as poorly differentiated adenocarcinoma. The pathological stages of the patients according to the current TNM classification were as follows: 3 patients with T2, 11 patients with T3, and 6 patients with T4 disease. There was no lymph node metastasis in 3 cases (N0). In addition, one colectomy case without lymph node metastasis was staged N1c due to TDs.
The distribution of 254 foci was as follows: 152 foci were Type A (59.8%), 74 foci were Type X (29.1%), and 28 foci were Type F (11%). Type A foci included 14 (9.2%) ampulla of Vater adenocarcinomas, 39 (25.7%) stomach adenocarcinomas (IMVI: n = 15 and EMVI: n = 24), 77 (50.7%) pancreas adenocarcinomas, and 22 (14.4%) colon adenocarcinomas (IMVI: n = 4 and EMVI: n = 16). Type X foci included 6 (8.1%) ampulla of Vater adenocarcinomas, 12 (16.2%) stomach adenocarcinomas, and 9 (12.2%) colon adenocarcinomas (IMVI: n = 3 and EMVI: n = 6). Type F included 9 (32.1%) stomach adenocarcinomas and 19 (67.9%) colon adenocarcinomas.
Comparison of immunohistochemical markers
Positive controls stained for all immunohistochemical markers were included in the analysis.
CD31 was negative in all foci [Figure 1]. Of note, the evaluation of CD31 was difficult due to all endothelial cells expressing CD31.
|Figure 1: An example of an extramural type of venous invasion is presented. A focus of a tumoral glandular structure is shown (a, H and E, magnification is ×100). CD31 is negative (b, magnification is × 100) and SMM is positive and the staining score is “+3” (c, magnification is × 100)|
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For all foci, the transgelin and SMM expression results are shown in [Table 2]. SMM was negative in 21 foci, and among these foci, 14 (66.7%) foci were positive for transgelin. The IS of the foci was 3+ for 10 foci (71.4%), 2+ for 3 foci (21.4%), and 1+ for 1 (7%) focus. SMM was positive in 2 (0.9%) foci (IS = 3+), and both foci were negative for transgelin. Transgelin staining was statistically more sensitive than SMM staining (P < 0.001).
|Table 2: Transgelin and smooth muscle myosin expression results for all types of focus|
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For Type A foci [Figure 2] and [Figure 3], both transgelin and SMM were positive in 141 foci (92.8%). There was no SMM staining in 9 foci, and 6 of the foci stained (66.7%) positive for transgelin (IS = 3+). Transgelin staining was statistically more sensitive than SMM staining (P = 0.001).
|Figure 2: An example of Type A focus from a gastric adenocarcinoma (a) H and E, (b) SMM, and (c) transgelin stain. The magnification is × 100 in these three microphotographs|
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|Figure 3: Two examples of Type A. Both of them are extramural type. (a) H and E, (b) SMM, and (c) transgelin stain. The magnification is ×40 in these three microphotographs of the first case. (d) H and E, (e) SMM stain, immunohistochemical score +2, (f) transgelin stain, immunohistochemical score +3. The magnification is ×100 in these three microphotographs of the second case|
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For Type X foci [Figure 4], transgelin was positive in all foci. Thus, the statistical evaluation could not be completed for Type X. SMM was negative in 2 (2.7%) foci (ISs were 1+ and 3+ for transgelin).
|Figure 4: Two examples of Type X. (a and d) H and E, there is not a clue for venous invasion, except eosinophilia around tumor nodule, (b and e) SMM, and (c and f) transgelin stain. The magnification is ×100 in these six microphotographs|
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For Type F foci [Figure 5], 4 (14.2) foci were negative for both transgelin and SMM. SMM was negative in 10 foci, and 6 of the Type F foci (60%) were positive for transgelin (ISs were 2+ for 3 foci and 3+ for the remaining foci). Transgelin staining was statistically more sensitive than SMM staining (P = 0.010).
|Figure 5: Two examples of type F and both of them were sampled as lymph node. (a and d) H and E, (b and e) SMM, and (c and f) transgelin stain. The magnification is ×40 in these six microphotographs|
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| Discussion|| |
In this study, we re-evaluated H and E-stained slides to identify “orphan arteriole” and “protruding tongue” indicators according to the literature, and we investigated the efficiency of smooth muscle markers (SMM and transgelin) and an endothelial marker (CD31) in IMVI, EMVI, and TDs in colorectal, gastric, and pancreatic adenocarcinomas. Our results showed that H and E staining is not adequate to demonstrate VI and that CD31 is not suitable because endothelial cells are destroyed when the tumor invades a vein. SMM and transgelin were first evaluated as positive (0) and negative (1). When transgelin and SMM were compared in all VI foci, transgelin was found to be a superior predictor of SMM (P < 0.001). Compared to SMM, transgelin was also a superior predictor of Type A and Type F foci (P = 0.001 and P = 0.10, respectively). In Type X foci, which were considered positive with any marker according to the definition, all were stained with transgelin, and 97.3% of the foci stained positive for SMM. Our data also showed that although transgelin is an experimental/research-based stain, it can be useful for routine practice. In addition, transgelin was superior to SMM in detecting VI and TD (P < 0.001 and P = 0.01 respectively). However, we could make no distinction as to whether the positive TDs represented metastatic lymph nodes or VI.
At the end of the 1930s, Brown and Warren reported that VI causes visceral metastases in colorectal cancers. Currently, although TNM is the most important prognostic criterion for tumors, VI is known to have prognostic significance in all tumors. Many studies on VI have been performed on CRC, and Stage III and Stage II CRC patients with high-risk factors undergo systemic adjuvant therapy. VI status is important for patients because it is considered one such high-risk factor.
In gastric adenocarcinomas, VI has been shown to correlate with low survival and tumor recurrence., In retrospective studies, Li et al. performed VI analysis in routine H and E-stained slides from 361 gastric carcinoma patients and showed that VI status was significantly correlated with lower disease-free survival and cancer-specific survival. Moreover, Setälä et al. showed that TNM was the most relevant factor and that the Lauren classification, vascular invasion, and lymphatic invasion of tumors were also independent prognostic factors for survival.
The literature contains many well-defined criteria for diagnosing VI, and numerous studies have investigated VI foci with histochemical markers and have demonstrated the superiority of these markers to H and E staining., However, we previously published that vessel invasion could be identified using a more sensitive method, including h-caldesmon and desmin stains, compared to elastin stains. Stewart et al. showed that the focus staining of VI in mucinous adenocarcinoma of the colon was 9% for elastin and up to 30% for h-caldesmon. In this study, among all VI foci, the most commonly detected focus type was “Type A,” suggesting that the appearance of an “orphan arteriole” is a specific finding for VI. Surprisingly, not only the suspect foci but also some of the VI foci that could not be recognized by H and E staining or predicted using existing criteria were observed with the immunohistochemical stains (Type X foci). When looking closely at these foci in H and E-stained slides, we observed evidence of vessel wall like eosinophilia surrounding the foci. Thus, these foci would have been missed without special staining methods because they are indistinguishable using an H and E stain.
EMVI has been shown to be an independent prognostic factor in many studies,, but there are only two studies showing that IMVI is a prognostic factor., In our study, 106 VI foci were located in the organ wall of 9 patients with colon and stomach cancer. Both IMVI and EMVI foci were detected in all the cases, with the exception of 1 rectum adenocarcinoma that received neoadjuvant chemotherapy.
Our study showed that CD31 was not as successful at detecting VI as transgelin and SMM, which may be attributed to the disappearance of endothelial cells in the initial tumor-infiltrating vein. Van Wyk et al. showed that elastin staining is a better method of detecting lymphatic or blood vessel invasion than CD31 or D2-40 staining and that VI is an independent prognostic factor in colorectal cancer. Using elastin, trichrome, CD31, and desmin staining to examine 115 cases of renal cell carcinoma, Bonsib and Bhalodia found that CD31 was negative in endothelial cells in intravenous growing and interstitial infiltrating tumors, and these authors suggested that the vessel wall may be visible by desmin and trichrome staining only in the invasion of intralobar and larger venules.
In the current edition of the TNM staging system, the value of TDs in the nodal stage remains under discussion., It is suggested that Stage II colon carcinomas with pN1c should be considered as Stage III and that TDs can be considered as positive lymph nodes. In our study, TDs showed positive findings, either partially or with both SMM and transgelin staining, but as the expression of smooth muscle markers was observed in the capsules of normal lymph nodes, no distinction could be made as to whether these foci were positive lymph nodes or VI. Nevertheless, given that the presence of VI or free TD leads to the application of adjuvant chemotherapy/radiotherapy in the form of pN1c in cases of lymph node metastasis, it is thought to be a valuable finding that SMM and/or transgelin positivity may be observed in a Type F focus (although VI/TD discrimination could not be determined).
SMM is routinely used to identify myoepithelial cells of smooth muscle cell origin in breast lesions, and transgelin is an experimental/research-based stain. Because transgelin was found to be a more sensitive marker than SMM in our study, we recommend its use for evaluating VI and TD. There are no studies in the English literature regarding the use of SMM or transgelin for the detection of VI. Although these markers have some limitations: (i) Because SMM and transgelin are smooth muscle markers, it is not possible to assess the foci of VI around the muscularis propria and muscularis mucosa. (ii) Positive staining was observed with both markers in the normal lymph node capsule. (iii) A normal smooth muscle layer can be observed around the pancreatic duct of Wirsung and large bile ducts, and nuclear atypia must be investigated before these areas are evaluated as VI. (iv) Because the follicular dendritic cell network with SMM can be positive, care should be taken when evaluating foci where lymphocytes and/or lymphoid follicles are present.
| Conclusion|| |
We examined foci thought to represent VI in colon, pancreas, and stomach adenocarcinomas and searched for improvements in the methods used to identify them. We showed that smooth muscle stains, such as SMM and transgelin, are superior to endothelial stains, such as CD31. However, more studies are needed to confirm these findings.
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Conflicts of interest
There are no conflicts of interest.
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Department of Pathology, Faculty of Medicine, Gazi University, Ankara 06560
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]