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Reappraisal of Serosal Invasion in Patients With T3 Colorectal Cancer by Elastic Stain: Clinicopathologic Study of 139 Surgical Cases With Special Reference to Peritoneal Elastic Lamina Invasion.

According to the World Health Organization classification of malignant tumors, pathologic stage pT3 colorectal cancer (CRC) is defined as a cancer invading through the muscularis propria into pericolorectal tissues, whereas pT4 CRC is defined as a cancer penetrating to the surface of visceral peritoneum. (1) The peritoneal, pleural, and pericardial cavities are lined by serosal membrane consisting of mesothelial cells, basement membrane, and submesothelial layer. (2) One of the unique histologic features of the serosal membrane is the presence of elastic lamina in the submesothelial layer, which can be visualized by elastic staining. Visceral pleural invasion is one of the most important prognostic factors in patients who undergo complete resection for non-small cell lung cancer. (3,4) Lung cancers with visceral pleural elastic lamina invasion are considered to be pT2 even when they are 3 cm or less in size, according to the TNM staging system for lung cancer. (5) However, there have been only a few previous reports on the clinicopathologic significance of peritoneal elastic lamina invasion (PELI) by CRC. (6-11) Although most of the previous studies demonstrated that pT3 tumors with PELI were associated with worse survival than pT3 tumors without PELI, the prognostic significance of PELI in pT3 tumors in the most recent 3 studies remains controversial. (9-11) Also, the number of sections per case stained for elastic lamina is different among the previous studies. (6-11) In this study elastic stain was applied to a single block containing the deepest tumor invasion, and the clinicopathologic importance of detecting tumor invasion beyond the peritoneal elastic lamina was determined. This allows one to stratify patients with pT3 CRC who might benefit from more aggressive therapy.

MATERIALS AND METHODS

One hundred thirty-nine consecutive in-house surgical specimens of pT3 CRC and 30 consecutive in-house surgical specimens of pT4a CRC obtained from patients who underwent surgery between 1993 and 2011 were reviewed in this study (Tables 1 and 2). Case selections for pT3 CRC were restricted to the cases with the sections containing the deepest adenocarcinoma invasion partially or entirely covered with the peritoneum. Patients who received preoperative chemotherapies and/or radiotherapies were excluded from this study. All the tumors included in this study were located on the proximal side of the peritoneal reflection, where the bowel is surrounded by the peritoneum. Clinicopathologic factors including patient's age and sex, location of the tumor, histologic grade, lymphovascular invasion, lymph node metastasis, recurrence, and survival were retrospectively reviewed. Tumor grade was categorized as low (well or moderately differentiated) or high (poorly differentiated). This study was approved by the institutional review board. Informed consent for the study was obtained by all participants before the procedure.

Elastic staining was performed on a single section of pT3 tumors containing the deepest tumor invasion using the elastic Van Gieson staining kit (HT25A, Sigma-Aldrich, Buchs, Switzerland). The elastic stain was not performed on pT4a cases because tumor cells are present at the serosal surface in all pT4a cases. Cases with tumor invasion beyond the peritoneal elastic lamina were designated as positive for PELI, and cases without tumor invasion beyond the peritoneal elastic lamina were designated as negative for PELI. The peritoneal elastic lamina was occasionally disrupted not only by tumor invasion, but also by tumor-associated inflammation or desmoplastic reaction. In such cases, a line between the remaining elastic lamina was drawn, and only cases with tumor invasion beyond the line were designated as positive for PELI.

The associations between the presence of PELI and clinicopathologic factors were examined. For categorical variables the [chi square] test and for continuous variables the Student t test was used. Analyses of overall and recurrence-free survival were performed according to the Kaplan-Meier method. The statistical significance of the differences in the survival distribution among the prognostic groups was evaluated by the log-rank test. Multivariate analysis was performed using the Cox proportional hazards model. Only variables with P values less than 0.1 remained in the final model. Data were analyzed with SAS 9.4 (SAS Institute Inc, Cary, North Carolina). The statistical difference was considered to be significant if the P value was below .05.

RESULTS

Elastic lamina in the submesothelial layer was visualized by elastic staining in all cases with similar intensity and continuity (Figure 1, A and B). Peritoneal elastic lamina invasion was often seen in the fibrotic areas of invasion front with peritoneal surface retraction (Figures 2 and 3). Peritoneal elastic lamina invasion was identified in 23.0% (32 of 139) of the pT3 CRC cases (Table 1). Peritoneal elastic lamina invasion was significantly associated with patient's age (P = .002), location of the tumor (P = .006), lymphovascular invasion (P < .001), lymph node metastasis (P < .001), and recurrence (P = .007). The overall and recurrence-free survival rates of patients positive for PELI were poorer than for patients negative for PELI, but were better than those for pT4a patients (P < .001 and P < .001, respectively) (Figures 4 and 5). Tables 3 and 4 show the results of the multivariate survival analysis. Patient age and the presence of recurrence were found to be independent overall prognostic factors (both P < .001), and the presence of recurrence was found to be an independent disease-free prognostic factor (P < .001).

COMMENT

In this study, the prognosis of patients positive for PELI was significantly poorer than that of those negative for PELI, but better than that of those with pT4a tumors. This clearly reflects the extent of tumor invasion, which was classified into 3 subgroups based on PELI: tumors without PELI, tumors with PELI, and tumors with penetration to the surface of visceral peritoneum, indicating that a sharp distinction can be made between pT3 tumors with PELI and those without PELI. In univariate analysis, PELI was significantly associated with lymphovascular invasion, lymph node metastasis, and recurrence. It is quite conceivable that those factors might have worked as confounders in multivariate analysis, and, therefore, multivariate analysis did not reveal that PELI was an independent poor prognostic factor. The prognostic analysis of patients with pT3 CRC subclassified into 2 groups clearly revealed that PELI can be useful for stratifying patients with pT3 CRC who might benefit from more aggressive therapy. A categorization of the primary lung cancer extension based on the cancerous involvement of elastic lamina has been established. (5) The colon and rectum have an anatomic analogy to the lung in that the colon and rectum above the peritoneal reflection are also surrounded by a subserosal elastic lamina. Because of this anatomic similarity to the lung, it is quite natural that PELI becomes a useful landmark for subdividing pT3 CRCs. Recent advances in molecular biology and genetics have created various new prognostic markers. Elastic stain is an easy and inexpensive staining method compared with those for molecular biological analysis, and can be performed at any facility. In a future revision of the TNM staging system for CRC, PELI should be considered for inclusion in the new TNM descriptions.

In this study, case selections for pT3 CRC were restricted to the cases with the sections containing the deepest adenocarcinoma invasion partially or entirely covered with the peritoneum. The peritoneum is histologically characterized by the presence of elastic lamina in the submesothelial layer. Therefore, an elastic lamina was found in all cases with similar intensity and continuity. Previous studies have reported that the frequencies of elastic lamina invasion in pT3 CRC were from 16.7% to 52.6%. (7-11) In this study, PELI was found in 23.0% (37 of 129) of the patients with pT3 CRC. Various studies have examined the differing number of sections per tumor stained for elastic lamina. Kojima et al (8) examined a mean of 4.6 sections per case (range, 1 to 18 sections), and found PELI in 248 out of 564 cases (44.0%), Yokota et al (11) examined a mean of 4.0 sections (range, 216), and found PELI in 182 out of 436 cases (41.7%), and Liang et al (10) performed only one elastic stain per case, selecting the sections that demonstrated the closest area of tumor invasion to the surface of the peritumoral adipose tissue, and found PELI in 60 out of 244 cases (24.6%). From a practical point of view, we performed only one elastic stain per case, and found a similar incidence of PELI to that observed in the study by Liang et al. (10) Differences in the number of sections stained may be associated with the relatively low positivity of elastic lamina invasion in this study. Three previous studies examined pT3 CRC including cases with positive lymph node metastasis, (7,8,11) whereas 2 other previous studies examined pT3 CRC without positive lymph node metastasis. (9,10) In this study, we examined pT3 CRC including cases with lymph node metastasis, and found significant association of PELI with lymphovascular invasion (P < .001) and lymph node metastasis (P < .001). Lymph node metastasis is an integral component of the staging system and an important prognostic marker in CRC. (1,6,7) The current study revealed that PELI is a significant predictive marker for lymphovascular invasion and lymph node metastasis.

The finding of a tumor with fibrous stroma and hemosiderin deposition beyond the elastic lamina might indicate that the tumor has perforated and healed by fibrosis. The implication of this type of local peritoneal reaction remains controversial. (12) The College of American Pathologists suggests the following findings to represent serosal involvement (pT4a): tumor present at the serosal surface with inflammatory reaction, mesothelial hyperplasia, and/or erosion/ulceration, and free tumor cells on the serosal surface with underlying ulceration of the visceral peritoneum. A mesothelial inflammatory and/or hyperplastic reaction with tumor close to, but not at, the serosal surface is currently not included in the pT4a category according to the College of American Pathologists cancer protocol. (13)

The tumor area beyond the peritoneal elastic lamina was reported to be enriched with tumor-budding foci and fibrosis, which may be associated with tumor progression and metastasis. (8) In this study, some of these findings such as tumor budding, desmoplastic stromal reaction, fibrosis, and inflammation in the tumor area beyond the peritoneal elastic lamina were found in all cases. The tumor area beyond the peritoneal elastic lamina is an intriguing microenvironment to investigate colon cancer progression and metastasis.

In summary, PELI was identified in 23.0% (37 of 139) of the pT3 CRC cases. Positive PELI is a significant predictive factor for lymph node metastasis and recurrence-free survival in patients with pT3 CRC, indicating that pT3 tumors with PELI should be treated like pT4a tumors.

CONCLUSION

Positive PELI is a significant predictive factor for lymph node metastasis and recurrence-free survival in patients with pT3 CRC, indicating that pT3 tumors with PELI should be treated like pT4a tumors.

References

(1.) Sobin L, Gospodarowicz M, Witterkind C, et al. TNM Classification of Malignant Tumours. 7th ed. Oxford, United Kingdom: John Wiley and Sons Ltd; 2009.

(2.) Carter D, True L, Otis CN. Serosal membranes. In: Mills SE, ed. Histology for Pathologists. 3rded. Philadelphia, PA: Lippincott Williams and Wilkins; 2007: 547-562.

(3.) Shimizu K, YoshidaJ, Nagai K, et al. Visceral pleural invasion isan invasive and aggressive indicator of non-small cell lung cancer. J Thorac Cardiovasc Surg. 2005; 130(1):160-165.

(4.) Osaki T, Nagashima A, Yoshimatsu T, et al. Visceral pleural involvement in nonsmall cell lung cancer: prognostic significance. Ann Thorac Surg. 2004; 77(5): 1769-1773.

(5.) Rami-Porta R, CrowleyJJ, Goldstraw P. The revised TNM staging system for lung cancer. Ann Thorac Cardiovasc Surg. 2009; 15(1); 4-9.

(6.) Katsumata D, Fukui H, Ono Y, et al. Depth of tumor invasion in locally advanced rectal cancer correlates with patient's prognosis: the usefulness of elastic stain for its measurement. Surg Today. 2008; 38(2):115-122.

(7.) Shinto Eiji, Ueno H, Hashiguchi Y, et al. The subserosal elastic lamina: an anatomic landmark for stratifying pT3 colorectal cancer. Dis Colon Rectum. 2004; 47(4):467-473.

(8.) Kojima M, Nakajima K, Ishii G, et al. Peritoneal elastic laminal invasion of colorectal cancer: the diagnostic utility and clinicopathologic relationship. Am J Surg Pathol. 2010; 34(9):1351-1360.

(9.) Grin A, Messenger D, Cook M, et al. Peritoneal elastic lamina invasion: limitations in its use as a prognostic marker in stage II colorectal cancer. Hum Pathol. 2013; 44(12):2696-2705.

(10.) Liang WY, Chang WC, Hsu CY, et al. Retrospective evaluation of elastic stain in the assessment of serosal invasion of pT3No colorectal cancer. Am J Surg Pathol. 2013; 37(10):1565-1570.

(11.) Yokota M, Kojima M, Nomura S, et al. Clinical impact of elastic laminal invasion in colon cancer: elastic laminal invasion-positive stage II colon cancer is a high-risk equivalent to stage III. Dis Colon Rectum. 2014; 57(7):830-838.

(12.) Frankel WL, Jin M. Serosal surfaces, mucin pools, and deposits, oh my: challenges in staging colorectal carcinoma. Mod Pathol. 2015; 28(suppl 1):S95-S108.

(13.) College of American Pathologists. Protocol for the Examination of Specimens From Patients With Primary Carcinoma of the Colon and Rectum. Northfield, IL: College of American Pathologists; 2012.

Yukihiro Nakanishi, MD, PhD; Charles LeVea, MD; Shiva Dibaj, MS; Fadi Habib, MD; Richard Cheney, MD; Kazunori Kanehira, MD

Accepted for publication May 11, 2015.

From the Departments of Pathology (Drs Nakanishi, LeVea, Habib, Cheney, and Kanehira) and Biostatistics and Bioinformatics (Ms Dibaj), Roswell Park Cancer Institute, Buffalo, New York; and the Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana (Dr Nakanishi).

The authors have no relevant financial interest in the products or companies described in this article.

Presented at the 2015 United States and Canadian Academy of Pathology Annual Meeting; March 24, 2015; Boston, Massachusetts.

Reprints: Yukihiro Nakanishi, MD, PhD, Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave, SL-79, Room 6520, New Orleans, LA 70112 (e-mail: ynakanis@tulane.edu).

Caption: Figure 1. Elastic lamina in the submesothelial layer is visualized by elastic staining (hematoxylin-eosin, original magnification X400 [A]; elastic stain, original magnification X400 [B]).

Caption: Figure 2. Peritoneal elastic lamina invasion was often seen in the fibrotic areas of invasion front with peritoneal surface retraction (elastic stain, original magnification X4).

Caption: Figure 3. High-power view of peritoneal elastic lamina invasion shows tumor budding and desmoplastic stromal reaction (elastic stain, original magnification X400).

Caption: Figure 4. The overall survival rates of patients positive for peritoneal elastic lamina invasion (PELI) were poorer than those of patients negative for PELI, but were better than those of pT4a patients (P <.001). Abbreviations: ELI, elastic lamina invasion; OS, overall survival.

Caption: Figure 5. The recurrence-free survival rates of patients positive for peritoneal elastic lamina invasion (PELI) were poorer than those of patients negative for PELI, but were better than those of pT4a patients (P < .001). Abbreviations: ELI, elastic lamina invasion; RF, recurrence-free.

Please Note: Illustration(s) are not available due to copyright restrictions.
Table 1. Associations Between Peritoneal Elastic Laminal Invasion
(PELI) and Clinicopathologic Factors of pT3 Colorectal Cancer

                                 PELI Positive   PELI Negative     P
                                   (n = 32)        (n = 107)

Sex, No. (%)                                                      .22
  Female                           11 (34.4)       50 (46.7)
  Male                             21 (65.6)       57 (53.3)
Age, median, (range), y          72.0 (34-91)    63.5 (30-83)     .002
Primary site, No. (%)                                             .006
  Right colon                      20 (62.5)       40 (37.3)
  Transverse colon                  1 (3.1)        16 (15.0)
  Left colon                        0 (0)           5 (4.7)
  Rectosigmoid                     11 (34.3)       46 (43.0)
Tumor differentiation, No. (%)                                    .19
  Low grade                        28 (87.5)      101 (94.4)
  High grade                        4 (12.5)        6 (5.6)
Positive LVI, No. (%)              14 (43.8)       17 (15.9)     <.001
Lymph node metastasis, No. (%)     28 (87.5)       39 (36.4)     <.001
Recurrence, No. (%)                 9 (28.1)       10 (9.3)       .007

Abbreviation: LVI, lymphovascular invasion.

Table 2. Clinicopathologic Features of pT4a
Colorectal Cancer

Features                         pT4a Colorectal Cancer
                                        (n = 30)
Sex, No. (%)
  Female                               15 (50.0)
  Male                                 15 (50.0)
Age, median (range), y               63.3 (32-88)
Primary site, No. (%)
  Right colon                          12 (40.0)
  Transverse colon                      9 (30.0)
  Left colon                            2 (6.7)
  Rectosigmoid                          7 (23.3)
Tumor differentiation, No. (%)
  Low grade                            22 (73.3)
  High grade                            8 (26.7)
Positive LVI, No. (%)                  11 (36.7)
Lymph node metastasis, No. (%)         21 (72.4)
Recurrence, No. (%)                    11 (36.7)

Abbreviation: LVI, lymphovascular invasion.

Table 3. Multivariate Analysis of Clinicopathologic
Features With Overall Survival

                             Hazard Ratio        P
Feature                    (95% Confidence
                              Interval)

Recurrence                10.96 (6.12-19.61)   <.001
Lymph node metastasis      4.71 (1.80-12.33)    .002
Age                        1.04 (1.02-1.07)    <.001
Lymphovascular invasion    0.64 (0.37-1.09)     .10
Positive PELI              0.71 (0.35-1.47)     .36

Abbreviation: PELI, peritoneal elastic laminal invasion.

Table 4. Multivariate Analysis of Clinicopathologic
Features With Disease-Free Survival

Feature                       Hazard Ratio
                            (95% Confidence      P
                               Interval)

Recurrence                62.10 (21.09-182.88)   <.001
Lymph node metastasis      7.98 (1.43-44.44)      .02
Lymphovascular invasion    0.60 (0.33-1.10)       .10
Positive PELI              0.93 (0.43-2.03)       .86

Abbreviation: PELI, peritoneal elastic laminal invasion.
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Author:Nakanishi, Yukihiro; LeVea, Charles; Dibaj, Shiva; Habib, Fadi; Cheney, Richard; Kanehira, Kazunori
Publication:Archives of Pathology & Laboratory Medicine
Date:Jan 1, 2016
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