The Clinical Significance of Elastic Lamina Invasion in Patients with pStage II Colorectal Cancer: A Notable Prognostic Indicator

doi:10.21203/rs.3.rs-4911917/v1

Background

Some colorectal cancers (CRCs) are clinically diagnosed as cT4a with serosal invasion (SI). However, the cT4a is most often underdiagnosed pathologically as pT3 without SI by hematoxylin-eosin (H&E) staining alone. Using Elastica-Van Gieson (EVG) staining, some pT3 tumors invade the elastic lamina (EL), which extends just below the serosal layer. Recently, EL invasion (ELI) has been described as a poor prognostic factor for disease-free (DFS) and overall survival (OS) in patients with pStage II CRC. However, its clinicopathological significance remains unclear due to the limited number of studies and poor understanding of ELI.

Objective

This study investigated the association between ELI and prognosis.

Methods

After 1982, pathological diagnosis was routinely performed using H&E and EVG staining methods and long-term follow up was performed until 2016. All clinicopathological features including ELI were prospectively registered into our computer and 605 patients with pStage II CRC were collected from the database. Based on ELI status, pT3 was divided into pathological three categories; pT3ELI − was defined as pT3a, pT3ELI + as pT3b and unidentified EL (pT3EL−) as pT3u.

Results

Using H&E staining alone, gross cT4a was most often pathologically underdiagnosed as pT3 (93.4%) and very rarely as pT4a, resulting in a large diagnostic discrepancy. Using EVG staining, 59.2% of cT4a cases were diagnosed as pT3b. The 10-year DFS and OS rates were similar for pT3a and pT3u. However, the 10-year DFS and OS rates of pT3b were significantly lower than that of pT3a (76.8% vs. 95.8%, p < 0.001 and 58.7% vs. 69.3%, p = 0.003, respectively) but did not differ from that of pT4a (74.3%, p = 0.771 and 51.5%, p = 0.157, respectively). Multivariate analysis identified ELI as the strongest independent risk factor for recurrence and CRC-specific death (p < 0.0001).

Conclusions

A better understanding of ELI allows us to reconsider the diagnostic discrepancy of serosal invasion, i.e., pT3b should be considered pT4a. The ELI-based subclassification of pT3 is expected to be incorporated into the TNM staging system in the future. ELI is a notable prognostic indicator in patients with pStage II CRC.

elastic lamina invasion

colorectal cancer

serosal invasion

survival

Colorectal cancer (CRC) is one of the most common cancers in the world [1]. Some CRCs are diagnosed clinically as cT4a with serosal invasion (SI). With hematoxylin-eosin (H&E) staining alone, the cT4a is most often underdiagnosed pathologically as pT3without SI, i.e., invasion into subserosa or non-peritonealized pericolic/perirectal tissue, and very rarely as pT4a with a high risk potential of peritoneal recurrence [2–5]. There is a large discrepancy in the diagnosis of SI [6]. The distinction between pT3 and pT4a continues to be a source of diagnostic confusion [7–13]. One of the reasons for the diagnostic difficulty appears to be diagnosis based on H&E staining alone, as pointed out by Lu [14]. However, using Elastica-Van Gieson (EVG) staining, some pT3 tumors invade the elastic lamina (EL), which extends just below the serosal layer. Therefore, some authors reported that pT3 with ELI + is considered occult pT4a [3, 8, 15]. In addition, Liang [16] proposed that pT3 with ELI + should be considered pT4a. Moreover, ELI + has been recently described as a poor prognostic factor in patients with pStage II colorectal cancer (CRC) [7, 17–20]. In lung cancer, the TNM staging system 7th edition [21] defined ELI as positive pleural invasion. Some studies have reported that ELI is associated with poor prognosis in lung cancer, gastric cancer [22–24], and pT3N0/N (+) CRC [7, 16–20, 25]. However, the TNM staging system 8th edition [26] has not yet defined ELI in CRC. This may be because the clinicopathological significance of ELI remains unknown. The pathological features of ELI were previously studied by the author (KS) and were briefly described in the research article published in 1984 [27]. On this occasion, the author reevaluated its clinicopathological significance and addressed issues that have long been questioned by surgeons. The present study improves the understanding of ELI and highlights the prognostic differences in ELI-based subclassification of pT3. It also discusses the diagnostic discrepancy between gross and pathological SI (cT4a/pT4a) from the perspective of the ELI.

Study Design and Inclusion/Exclusion Criteria

This is a retrospective cohort study. Data were collected from the computerized original database initiated by the author (KS) in 1982 [28–32]. The prospectively registered records from January 1982 to December 2013 were reviewed. A total of 3364 patients with CRC underwent surgical treatment. Among them, 605 patients with pStage II (pT3/pT4a) had a primary CRC with curative surgery. All tumors were located proximal to the peritoneal reflection, where the intestine is surrounded by the serosal membrane. Patients with multiple primary cancers, those who received neoadjuvant chemotherapy, and those who died in the hospital were excluded.

Tumor Location and Surgical Procedures

There were 209 cases in the right colon, 314 in the left colon and 82 in the intraperitoneal rectum. In accordance with Japanese clinical practice guidelines [33], D3 lymph node (LN) en-bloc dissection [34] was mainly performed in 504 patients, but D2/D1 LN dissection was selected in 101 patients depending on preoperative health status. Prior to 1995, 579 patients underwent open surgery. Subsequently, 26 patients underwent laparoscopic surgery.

Processing of Resected Specimens

The processing of resected specimens has been reported on previously [28–32]. Briefly, the serosal surface of fresh resected specimens was carefully observed to diagnose cT3 or cT4a. The mesenteric LNs were harvested immediately after surgery. Pericolic/perirectal LNs close to the main tumor were never harvested to avoid damaging the serosal membrane and extramural adipose tissue. After formalin fixation for 1 week, the entire tumor mass, including the serosa and extramural adipose tissue, was cut into large step-shaped sections. The blocks were then embedded in paraffin after alcohol dehydration. Thin 4–5 µm sections were cut from the paraffin-embedded blocks and routinely stained with both H&E and EVG staining methods.

Typical ELI and Definition of pT3 Classification

The author (KS) was responsible for the final pathological diagnosis of each patient from 1982 until 2014, when he retired. All pathological findings including ELI were sketched in detail on ISO B4 size paper as previously reported [32]. The elastic lamina (EL) is occasionally breached or disrupted by tumor or desmoplastic reaction, but can be traced by identifying fragments of elastic fibers. A typical ELI is shown in Fig. 1. ELI − indicates that the tumor invasion does not exceed the EL, and ELI + indicates that the tumor invasion exceeds the EL, while EL − indicates that the EL is unidentified. pT3 was sub-classified into three groups; pT3ELI − was defined as pT3a (Fig. 1a), pT3ELI + as pT3b (Fig. 1b), and pT3EL − as pT3u (Fig. 1c). pT4a was defined as cancer cells exposed to the serosal surface or in the peritoneal cavity (Fig. 1d), according to the TNM staging system 8th edition [26].

Postoperative Adjuvant Chemotherapy

Postoperative adjuvant chemotherapy with oral 5-Fu-based regimens was administered to 250 patients (41.3%).

Follow-up Examination

The date of the outpatient visit was recorded as the date of survival confirmation. The routine program included serum tumor marker measurements, chest radiography and abdominal ultrasound every three months for the first three years, every six months for the next two years and annually thereafter. When recurrence was suspected, a definitive diagnosis was made using computed tomography and/or magnetic resonance imaging. Local recurrence was defined as tumors occurring in the peritoneal cavity or pelvis within the field of the initial surgery. Distant metastases were defined as the tumors observed in the liver, lung, and other hematogenous organs. Other recurrences included peritoneal dissemination and intra/extra-abdominal LNs metastases. Follow-up began in 1983 and was performed annually [28–32]. The author (KS) was involved in the follow-up work until the end of September 2016.

Statistical Analysis

Statistical analyses were performed using StatView software (version 5.0J; SAS Institute, Inc. Cary NC, USA). Pearson's chi-square (χ²) test and logistic regression analyses were performed to estimate correlations and the risks of recurrence for categorical variables. Univariate and multivariate Cox’s regression analyses were performed to identify independent prognostic factors. The Kaplan-Meier method was used to calculate time-dependent survival probabilities. Log-rank (LR) and Breslow-Gehan-Wilcoxon (BGW) tests were performed to estimate the survival differences. All statistical tests were two-tailed, and p < 0.05 was considered statistically significant, with confidence intervals (CI) at the 95% level for hazard ratio (HR) and odds ratio (OR).

Survival Analyses

Patients lost to follow-up were treated as CRC-related deaths. Disease-free survival (DFS) was defined as the time from the date of surgery to the date of the first confirmed tumor recurrence or CRC-related death. Overall survival (OS) was defined as the time from the date of surgery to the date of death from any cause. All deaths were considered events.

Identification of EL and ELI

A total of 5205 tumor block sections of the resected specimens were obtained from 605 patients (mean 8.6, standard deviation [SD] 3.2, range 4–20, median 8.0). The EL was identified in 91.6%, and was unidentified in 8.4% (Tables 1a, b). The EL was well identified in sigmoid colon (S) and recto-sigmoid colon (Rs) cancers. The EL was not identified in 8–9% of cecum (C) and transverse colon (T) cancers, and was difficult to identify in 10–15% of ascending colon (A), descending colon (D), and intraperitoneal rectal (Ra) cancers (Table 1a). The combined categories A + D + Ra and C + T + S + Rs were significantly correlated with the frequency of EL identification (p = 0.021). In addition, the EL was not identified in a significantly higher rate of 32.7% in tumors < 3 cm in size (p < 0.0001, Table 1b). Regarding the circumferential distribution in A, D and Ra cancers, the unidentified rate was significantly higher in the posterior wall tumor (59.3%) than in the other circumferential distributions (p < 0.0001, Table 1c). As shown in Table 1d, ELI + was detected in 47.2% (272/576) of pT3 cases and in 100% (29/29) of all pT4a cases. ELI − was detected in 43.9% (253/576) of pT3 cases. A significant correlation was found between cT and pT categories (p < 0.0001).

Table 1

Identification of Elastic Lamina (EL) and Elastic Lamina Invasion (ELI)
a. EL and Tumor Location
EL		Tumor Location
EL	C	A	T	D	S	Rs	Ra	p-value	Total
identified	30	93	66	34	168	93	70	0.021*	554 (91.6)
unidentified (%)	3 (9.1)	11 (10.6)	6 (8.3)	4 (10.5)	9 (5.1)	6 (6.1)	12 (14.6)		51 (8.4)
C: cecum; A: ascending colon; T: transverse colon; D: descending colon; S; sigmoid colon; Rs: recto-sigmoid colon; Ra: intraperitoneal rectum above the peritoneal reflection; *A/D/Ra vs. C/T/S/Rs: Chi-square test showed a significant correlation between tumor location and EL.

b. EL and Tumor Size
EL	Tumor size
EL	< 3cm	3-5cm	≥ 5cm	Unknown	p-value	Total
identified	35	205	311	3	< 0.0001^*	554 (91.6)
unidentified (%)	17 (32.7)	29 (12.4)	5 (1.6)	0		51 (8.4)
^* Chi-square test showed a significant correlation between tumor size and EL.

c. EL and Circumferential Distribution at A, D, and Ra
EL	Circumferential Distribution
EL	Posterior	Anterior	Lateral	Whole circumference	Unknown	p-value	Total
identified	20	62	18	95	2	< 0.0001^*	197 (87.9)
unidentified (%)	16 (59.3)	4 (14.8)	5 (18.5)	2 (7.4)	0		27 (12.1)
A: ascending colon; D: descending colon; Ra: intraperitoneal rectum above the peritoneal reflection; ^* Chi-square test showed a significant correlation between circumferential distribution and EL.

d. ELI and cT
cT	pT3, n = 576, (%)			pT4a, n = 29, (%)	p-value	Total
cT	pT3a [ELI−]	pT3u [EL−]	pT3b [ELI+]	[ELI+]	p-value	Total
cT3 cT4a	145 (50.2) 108 (34.2)	51 (17.6) 0	85 (29.4) 187 (59.2)	8 (2.8) 21 (6.6)	< 0.0001^*	289 316
Total	253	51	272	29		605
^* Chi-square test showed a significant correlation between cT and pT categories.

TABLE 2. Postoperative Recurrence
a. Recurrence in each pT Category
pT	Recurrence			Logistic Regression Analysis for Recurrence
pT	Presence n (%)	Absence n (%)	Total	OR (95%CI)	p-value
pT3a (EL−)	11 (4.3)	242 (95.7)	253	1 (reference)	-
pT3u (EI−)	3 (5.9)	48 (94.1)	51	1.38 (0.370-5.115)	0.6347
pT3b (ELI+)	60 (22.1)	212 (77.9)	272	6.23 (3.190-12.154)	<0.0001
pT4a (ELI+)	7 (24.1)	22 (75.9)	29	7.00 (2.466-19.872)	0.0003
Total	79 (13.1)	526 (86.9)	605	-	-
ELI−: negative elastic lamina invasion; EL−: unidentified elastic lamina; ELI+: positive elastic lamina invasion; OR: Odds Ratio; Recurrence and pT category were significantly correlated (p<0.0001, χ²test). The rate of recurrence did not differ between pT3a and pT3u and between pT3b and pT4a. ORs were significantly higher for 3b and pT4a than for pT3a and pT3u.

b. First Recurrence Sites, including Multiple Sites of Recurrence
pT	Liver	Lung	Peritoneum	Local	Bone	Others	Total
pT3a (ELI−), (%)	4 (36.4)	4 (36.4)	1 (9.1)	2 (18.2)	0	0	11
pT3u (EI−), (%)	3 (100)	0	0	0	0	0	3
pT3b (ELI+), (%)	29 (40.8)	10 (14.1)	11(15.5)	10 (14.1)	2 (2.8)	9 (12.7)	71
pT4a (ELI+), (%)	3 (25.0)	2 (16.7)	3 (25.0)	2 (16.7)	1 (8.3)	1 (8.3)	12
Total (%)	39 (40.2)	16 (16.5)	15 (15.5)	14 (14.4)	3 (3.1)	10 (10.3)	97
ELI−: negative elastic lamina invasion; EL−: unidentified elastic lamina; ELI+: positive elastic lamina invasion; To avoid statistical bias caused by the diversity of the sites, associations between the site of recurrence and pT category were not analyzed.

c. Distant Metastasis and Peritoneal Recurrence
pT	Distant Metastasis			Peritoneal Recurrence
pT	Presence	Absence	Total	Presence	Absence	Total
pT3a (EL−), (%)	8 (72.7)	3 (27.3)	11	1 (9.1)	10	11
pT3u (EI−), (%)	3 (100)	0 (0)	3	0 (0)	3	3
pT3b (ELI+), (%)	41 (66.1)	30 (42.3)	71	11 (15.5)	60	71
pT4a (ELI+), (%)	6 (54.5)	6 (45.5)	12	3 (25.0)	9	12
Total (%)	58 (66.7)	39 (40.2)	97	15 (15.5)	82 (84.5)	97
ELI−: negative elastic lamina invasion; EL−: unidentified elastic lamina; ELI+: positive elastic lamina invasion; Distant metastasis was found at a high rate in each ELI-based pT category without correlation (p=0.3360, χ²test). Peritoneal recurrence occurred significantly more often in patients with pT3b and pT4a than in those with pT3a and pT3u (p<0.0001, χ²test).

Gross and Pathological Findings of Resected Specimens

Fresh resected specimens are shown in Figs. 2a-c. Gross findings of the serosa were unequivocal cT4a. As previously reported [13], the pathological diagnoses by H&E staining were pT3, pT3, and pT4a, respectively (Figs. 2d-f), whereas the diagnoses by EVG staining were pT3a/ELI−, pT3b/ELI+, and pT4a/ELI+, respectively (Figs. 2g-i). In the cases in Figs. 2a and 2b, the gross and pathological diagnoses were not concordant, but in the case of Fig. 2c, the diagnoses were compatible. This study included 316 cases of cT4a (Table 1d). Of these, 295 cases were diagnosed as pT3 (93.4%) and 21 cases (6.6%) were diagnosed as pT4a by H&E staining alone. There was a large discrepancy between the gross and pathological diagnoses of SI. Based on ELI status, 108 cases (34.2%) were diagnosed as pT3a and 187 cases (59.2%) were diagnosed as pT3b.

Causes of Death during Long-term Follow-up

The last survey for surviving patients was completed at the end of September 2016. Eligible surviving and censored patients were followed up for a mean of 126 months (range, 2–351, SD 73, median 114 months). Of these patients, 50 (9.4%) were followed up for less than 3 years, 56 (10.5%) for 3–5 years, 172 (32.3%) for 5–10 years, 216 (40.6%) for 10–20 years, and 38 (7.1%) for more than 20 years. Fourteen patients (2.3%) were lost to follow-up between 5 and 70 months after surgery. A total of 135 patients had died of malignant disease by the end of September 2016. Of these, 73 patients (54.1%) died of primary CRC. The remaining 62 patients died mainly from second primary malignancies of the liver, stomach, and lung. Regarding non-malignant disease, 137 patients died mainly from senility/dementia, cardiovascular, cerebrovascular and pulmonary diseases.

Postoperative Recurrence

Postoperative recurrence occurred in 79 (13.1%) of 605 patients (Table 2a). Recurrence and pT category were significantly correlated (p < 0.0001, χ² test). The rate of recurrence did not differ between pT3a and pT3u (4.3% vs. 5.9%, respectively) and between pT3b and pT4a (22.1% vs. 24.1%, respectively). Logistic regression analysis revealed significantly higher ORs for pT3b and pT4a than for pT3a and pT3u (p < 0.0001, OR: 6.23, 95%CI: 3.190-12.154 and p = 0.0003, OR: 7.00, 95%CI: 2.466–19.872, respectively). Among these, recurrence sites were found in 97 lesions including the liver and lung, followed by the peritoneum, local sites and others (Table 2b). To avoid statistical bias caused by the diversity of the sites, associations between the site of recurrence and pT category were not analyzed. Distant metastasis was found at a high rate in each ELI-based pT category without correlation (p = 0.3360, Table 2c). However, peritoneal recurrence occurred significantly more often in patients with pT3b and pT4a (15.5% and 25.0%, respectively) than in those with pT3a and pT3u (9.1% and 0%, respectively, p < 0.0001).

Survival Results

The 10-year DFS did not differ between pT3a and pT3u (95.8% vs. 94.0%, respectively; p = 0.5364, Fig. 3a). The 10-year OS also did not differ between the two categories (69.3% vs. 61.4%, respectively; p = 0.8268, Fig. 3b). However, as shown in Fig. 3c, patients with pT3b had a lower 10-year DFS than those with pT3a (76.8% vs. 95.8%, respectively; p < 0.0001) but were similar to those with pT4a (76.8% vs. 74.3%, respectively; p = 0.7710). They also had a lower OS than those with pT3a (58.7% vs. 69.3%, respectively; p = 0.0034, Fig. 3d), but were similar to those with pT4a (58.7% vs. 51.5%, respectively; p = 0.1574).

Univariate and Multivariate Analyses

Univariate analysis indicated tumor budding, perineural invasion, tumor deposit, and ELI were significant risk factors for postoperative recurrence (Table 3a). Following multivariate analysis, ELI was extracted as the strongest independent risk factor (p < 0.0001, HR: 4.78, 95%CI: 2.652–8.608). Similarly, these factors were significant predictors of CRC-specific death (Table 3b), and on multivariate analysis, ELI emerged as the strongest independent predictor (p < 0.0001, HR: 6.10, 95%CI: 3.090-12.033).

TABLE 3. Univariate and Multivariate Cox’s Regression Analysis for Independent Prognostic Factors
a. Postoperative Recurrence in pT3 Diseases
Independent variable		Univariate analysis HR (95%CI)	p-value	Multivariate analysis HR (95%CI)	p-value
Histology	well others	1 (reference) 1.36 (0.840-2.184)	0.2126	−	−
Lymphatic invasion	no yes	1 (reference) 1.64 (0.899-2.978)	0.1070	−	−
Venous invasion	no yes	1 (reference) 1.24 (0.501-3.082)	0.6384	−	−
Tumor budding	no yes	1 (reference) 1.79 (1.052-3.044)	0.0318	1 (reference) 1.33 (0.769-2.301)	0.3069
Perineural invasion	no yes	1 (reference) 3.34 (1.448-7.695)	0.0047	1 (reference) 1.57 (0.637-3.855)	0.3281
Tumor deposit	no yes	1 (reference) 3.64 (1.748-7.593)	0.0006	1 (reference) 2.21 (0.994-4.917)	0.0517
ELI	negative positive	1 (reference) 5.24 (2.925-9.368)	< 0.0001	1 (reference) 4.78 (2.652-8.608)	< 0.0001
ELI: elastic lamina invasion, HR: hazard ratio; CI: confidence interval; Multivariate analysis identified ELI as the strongest independent risk factor for postoperative recurrence.

b. CRC-specific Death in pT3 Diseases
Independent variable		Univariate analysis HR (95%CI)	p-value	Multivariate analysis HR (95%CI)	p-value
Histology	well others	1 (reference) 1.33 (0.803-2.212)	0.2663	−	−
Lymphatic invasion	no yes	1 (reference) 1.69 (0.906-3.165)	0.0989	−	−
Venous invasion	no yes	1 (reference) 1.47 (0.591-3.664)	0.4064	−	−
Tumor budding	no yes	1 (reference) 1.88 (1.084-3.270)	0.0247	1 (reference) 1.38 (0.781-2.436)	0.2679
Perineural invasion	no yes	1 (reference) 3.53 (1.522-8.162)	0.0033	1 (reference) 1.58 (0.639-3.894)	0.3232
Tumor deposit	no yes	1 (reference) 3.94 (1.882-8.264)	0.0003	1 (reference) 2.31 (1.033-5.160)	0.0414
ELI	negative positive	1 (reference) 6.75 (3.444-13.234)	< 0.0001	1 (reference) 6.10 (3.090-12.033)	< 0.0001
CRC: colorectal cancer; ELI: elastic lamina invasion; HR: hazard ratio; CI: confidence interval; Multivariate analysis identified ELI as the strongest independent predictor for CRC-specific death.

This retrospective cohort study clarified clinical and pathological issues, i.e., diagnostic discrepancy between cT4a and pT4a, and demonstrated the close relationship between ELI and prognosis in patients with pStage II CRC.

First of all, the serosal surface of fresh resected specimens should be carefully examined [8]. The serosal surface of cT4a often appears as whitish stripes/streaks, flattened indentation, or deep depression in fresh resected specimens [8, 15, 35] (Fig. 2a-c). Microscopically, desmoplastic reaction, isolated small glandular foci, and budding cells are usually observed on the tumor invasion front. Puppa [3] reported that when some advanced CRCs invade the serosa, the cap of fibrosis caused by a fibro-inflammatory reaction on the invasive front of the carcinoma is likely to replace areas of earlier peritoneal invasion. Such serosal findings are confidently diagnosed as cT4a with SI by many surgeons at the time of surgery or on a fresh resected specimen, whereas the cT4a is most often underdiagnosed as pT3 without SI according to the TNM classification [26]. Many surgeons are surprised by this under-diagnosis. Some authors have pointed out the significant discrepancy between the gross and pathological diagnoses of SI [3, 6]. This diagnostic discrepancy may be due not only to diagnostic ambiguity based on H&E staining alone [13, 14, 36], but also to differences in interpretation of the clinical and pathological definition of SI. The reported frequency of pT4a varies widely, ranging from 13.4–67.7% among 8 Japanese institutions [37] and the diagnostic difficulties are similar to those in other countries [9, 10]. On the other hand, when using EVG staining, some pT3 tumors invaded EL (Fig. 2h). However there were differences in EL identification according to tumor location and size (Tables 1a, b). Moreover, when the tumor is confined to the posterior wall, it may be difficult to identify the EL in ascending/descending colon cancer and intraperitoneal rectal cancer (Table 1c). Some authors reported that EL was detected less frequently in right-sided tumors than in left-sided tumors [6, 38, 39], which is similar to the results found in our study. Therefore, the likelihood of EL involvement depends not only on the degree of extramural invasion, but also on the tumor location, tumor size, and circumferential distribution [40]. This problem is also more likely to occur when the number of tumor block sections is small, because the serosa may not be included. In the present study, a mean of 8.6 block sections were stained with both H&E and EVG. Lu [14] also reported that the rate of unidentified EL was 18.5%, with a mean of 3.2 sections with EVG staining. Liang [18] used only 1 EVG section per case and reported that the EL could not be identified in 58% of cases. Their unidentified rates were higher than that of ours (8.4%, Tables 1a, b). In particular, small tumors < 3 cm should be carefully sectioned due to the high incidence of unidentified EL (32.7%, Table 1b). We believe that at least 4–5 block sections including the serosal layer would be required to identify the EL.

Regarding ELI, previous studies have reported that the frequency of pT3b ranged from 16.7–52.6% [7, 17–20, 38]. Our results showed a similar frequency of 47.2% (272/576, Table 1d) as described above. In addition, as shown in Figs. 1b and 2h, the majority of cT4a cases were diagnosed as pT3b with ELI+ (59.2%, Table 1d). When pT3 tumors closely infiltrate the serosal surface beyond the EL, even if a desmoplastic reaction occurs, there may be a moment when cancer cells are exfoliated into the peritoneal cavity [15]. Serosal cytologic smears demonstrated cancer cells in 19–26% of pT3 tumors [40, 41]. In other words, pT3b may be occult pT4a [3, 8, 15]. Liang [16] suggested that if ELI is present, the tumor should be considered SI positive and the stage should be pT4a. We agree with his opinion. This idea would resolve the diagnostic discrepancy between gross and pathological SI. EVG staining may be the best way to eliminate this discrepancy.

The oncological significance of ELI has been reported to be closely associated with prognosis in lung or gastric cancer [22–24]. Some studies have also reported that ELI is a poor prognostic factor for pT3 CRC [7, 16–20, 25]. Conversely, Grin [38] reported that ELI was not a negative prognostic factor in pStage II CRC. The small number of cases may also have contributed to this difference. Liang [18] pointed out that some previous studies had included pStage II and III disease, which did not show uniformity and introduced bias [7, 14, 16, 17, 19]. The present study specifically focused on patients with pStage II CRC to avoid any bias caused by different pStages [18, 38]. In addition, Japanese D3 LN en-bloc dissection [34], which is equivalent to complete mesocolic or total mesorectal excision [42, 43], appears to improve prognostic outcomes in patients with pT3a and pT3u. However, patients with pT3b and pT4a had a higher risk of recurrence than those with pT3a and pT3u (p < 0.0001, OR: 6.23 and p = 0.003, OR: 7.00, respectively, Table 2a). ELI + had a strong effect on postoperative recurrence [7, 17, 18]. The top four sites of first recurrence were the liver, lung, peritoneum, and local site (Table 2b), similar to other studies [7, 17, 19]. However, there are few reports on the association between distant metastasis and ELI [7, 17, 44]. Shinto [17] and Kojima [7] reported that distant metastasis occurred more frequently in the pT3b and pT4a groups than in the pT3a group. El-Aziz [44] also reported that distant metastasis rates differed significantly according to ELI status. However, in the present study, distant metastasis and ELI-based pT category were not associated (p = 0.3360, Table 2c). On the other hand, pT4a, i.e., pathological SI, is known to be a marker for predicting peritoneal recurrence. [3–5]. It is easy to understand that pT4a is prone to peritoneal dissemination, because cancer cells are exposed to the serosal surface and/or exfoliated into the peritoneal cavity. To our knowledge, there are few reports on the association between ELI and peritoneal dissemination. The present study showed that peritoneal recurrence was more strongly associated with pT3b and pT4a than with pT3a and pT3u (p < 0.0001, Table 2c), similar to the results of other studies [7, 20]. We can better understand the idea that pT3b may be occult pT4a [3, 8, 15].

In addition to the effect of D3 dissection, both pT3a and pT3u tumors appear to be low malignant, and the prognosis appears to be favorable [14, 18] (Figs. 3a, b). Conversely, the malignant potential of pT3b is considered to be much higher than that of pT3a/pT3u and very similar to that of pT4a [7, 17–20] (Figs. 3c, d). pT3b tumors appear to be a heterogeneous entity of pT3. Therefore, pT3b disease should be upgraded to pT4a disease and intensive adjuvant chemotherapy is recommended. However, some authors [38, 45] have found no prognostic significance between pT3a and pT3b in the DFS and OS, which differed from the results of our study (Figs. 3c and d).

According to univariate and multivariate analyses, ELI + was found to have a strong effect on recurrence and CRC-specific death in patients with pT3 disease (Tables 3a, b). Other studies have also shown that ELI + is an independent prognostic factor for postoperative survival [17] and is an independent risk factor for recurrence [7]. ELI appears to be a better prognostic indicator than traditional prognostic factors such as venous [28], lymphatic [29], and perineural invasion [30] and others. In patients with pStage II disease, ELI is very useful for identifying high-risk individuals, and confirming the diagnosis of SI. EVG staining is needed for identifying ELI.

In conclusion, a better understanding of ELI allows us to reconsider the diagnostic discrepancy of serosal invasion, i.e., pT3b should be considered pT4a. The ELI-based subclassification of pT3 is expected to be incorporated into the TNM staging system in the future. ELI is a notable prognostic indicator in patients with pStage II CRC.

CRC: Colorectal cancer; EL: elastic lamina; ELI: elastic lamina invasion, H&E: hematoxylin- eosin; EVG: Elastica-Van Gieson; DFS: disease-free survival; OS: overall survival; SI: serosal invasion; LN: lymph node; 5-Fu: fluorouracil; LR: log-rank; BGW: Breslow-Gehan-Wilcoxon; CI: confidence interval; HR: hazard ratio; OR: odds ratio; SD: standard deviation.

Ethics approval and consent to participate

In this study, retrospective anonymized clinicopathological data collected from computer were employed. This study was exempted from the need for informed consent and approved by the Ethics Committee of the Kurume University School of Medicine (Approval No 21187).

Consent for publication

Not applicable.

Availability of data and materials

The datasets generated and analyzed during the current study are not publicly available (due to the reason that another project by our team is ongoing using the same dataset), but are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

All authors have not received any external funding.

Authors’ Contributions

KS was responsible for obtaining and analyzing data, drafting manuscripts, and making critical revisions. KS and TH contributed to the pathological analyses and evaluations. FF participated in the drafting of the manuscript and development of study concept and design. TH, TY and KK were responsible for patient follow-up and related the collection of clinicopathological information. All authors contributed to the critical review of this manuscript and approved its submission.

Acknowledgement

The present study was supported by colorectal surgeons. They were involved in the surgery, long-term follow-up, and processing of resected specimens.

Details of corresponding author

Kazuo Shirouzu, Professor Emeritus of Kurume University School of Medicine (KUSM) (April 2014-present), Department of Pathology, KUSM (August 1980-November 1984), Professor and Chairperson of the Department of Surgery, KUSM (August 1995-March 2014). Department of Surgery, Japan Community Healthcare Organization, Kurume General Hospital (April 2014-present)

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No competing interests reported.

The Clinical Significance of Elastic Lamina Invasion in Patients with pStage II Colorectal Cancer: A Notable Prognostic Indicator

Status:

Journal Publication

Version 1

Abstract

Background

Objective

Methods

Results

Conclusions

Figures

Introduction

Methods

Study Design and Inclusion/Exclusion Criteria

Tumor Location and Surgical Procedures

Processing of Resected Specimens

Typical ELI and Definition of pT3 Classification

Postoperative Adjuvant Chemotherapy

Follow-up Examination

Statistical Analysis

Survival Analyses

Results

Identification of EL and ELI

Gross and Pathological Findings of Resected Specimens

Causes of Death during Long-term Follow-up

Postoperative Recurrence

Survival Results

Univariate and Multivariate Analyses

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1