Management of T1 colorectal cancers after endoscopic treatment based on the risk stratification of lymph node metastasis

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1 bs_bs_banner doi: /jgh GASTROENTEROLOGY Management of T1 colorectal cancers after endoscopic treatment based on the risk stratification of lymph node metastasis Hideyuki Miyachi,* Shin-ei Kudo,* Katsuro Ichimasa,* Tomokazu Hisayuki,* Hiromasa Oikawa,* Shingo Matsudaira,* Yuta Kouyama,* Yui Jennifer Kimura,* Masashi Misawa,* Yuichi Mori,* Noriyuki Ogata,* Toyoki Kudo,* Kenta Kodama,* Takemasa Hayashi,* Kunihiko Wakamura,* Atsushi Katagiri,* Toshiyuki Baba,* Eiji Hidaka,* Fumio Ishida,* Kenichi Kohashi and Shigeharu Hamatani*, *Digestive Disease Center, Showa University Northern Yokohama Hospital, Yokohama, Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, and Department of Pathology, The Jikei University School of Medicine, Tokyo, Japan Key words lymph node metastasis, muscularis mucosae, risk factor, T1 colorectal cancer. Accepted for publication 30 November Correspondence Dr Shin-ei Kudo, Digestive Disease Center, Showa University Northern Yokohama Hospital, 35-1 Chigasaki Chuo, Tsuzuki-ku, Yokohama City, Kanagawa , Japan. Declaration of conflict of interest: None. Guarantor of the article: Shin-ei Kudo, MD, PhD A summary of this study was presented at Digestive Disease Week, Chicago, USA, 5 May 2014 (oral presentation). Abstract Background and Aim: Recent advances in endoscopic technology have allowed many T1 colorectal carcinomas to be resected endoscopically with negative margins. However, the criteria for curative endoscopic resection remain unclear. We aimed to identify risk factors for nodal metastasis in T1 carcinoma patients and hence establish the indication for additional surgery with lymph node dissection. Methods: Initial or additional surgery with nodal dissection was performed in 653 T1 carcinoma cases. Clinicopathological factors were retrospectively analyzed with respect to nodal metastasis. The status of the muscularis mucosae (MM grade) was defined as grade 1 (maintenance) or grade 2 (fragmentation or disappearance). The lesions were then stratified based on the risk of nodal metastasis. Results: Muscularis mucosae grade was associated with nodal metastasis (P = 0.026), and no patients with MM grade 1 lesions had nodal metastasis. Significant risk factors for nodal metastasis in patients with MM grade 2 lesions were attribution of women (P = 0.006), lymphovascular infiltration (P < 0.001), tumor budding (P = 0.045), and poorly differentiated adenocarcinoma or mucinous carcinoma (P = 0.007). Nodal metastasis occurred in 1.06% of lesions without any of these pathological factors, but in 10.3% and 20.1% of lesions with at least one factor in male and female patients, respectively. There was good inter-observer agreement for MM grade evaluation, with a kappa value of Conclusions: Stratification using MM grade, pathological factors, and patient sex provided more appropriate indication for additional surgery with lymph node dissection after endoscopic treatment for T1 colorectal carcinomas. Introduction With the recent advances in endoscopic treatment, many T1 colorectal carcinomas are now resected endoscopically with negative margins, 1 and the proportion of early carcinomas amenable to endoscopic resection has increased. 2 After endoscopic treatment, however, additional surgery with lymph node dissection should be considered if there is a risk of lymph node metastasis (LNM), and it is therefore important to elucidate the risk factors for LNM in T1 carcinoma patients. Previously identified risk factors for LNM in T1 carcinoma include lymphovascular infiltration, histological grade, tumor budding, and degree of submucosal invasion. 3 5 However, most previous studies were based on small cohorts, 2 and only a few took into consideration pit pattern, morphology, location, growth type, status of the muscularis mucosae (MM), desmoplastic reaction (DR), or absence of background adenoma (BGA). 6 8 Degree of submucosal invasion is a commonly used risk factor and is broadly defined by its vertical depth or horizontal width. Although initial studies in the 1990s focused on the horizontal extent of invasion or the status of the MM, 6,9 13 these have been considered less important than the vertical depth. According to the National Comprehensive Cancer Network guidelines, the criteria for curative endoscopic resection for T1 carcinomas are negative margins, 1126 Journal of Gastroenterology and Hepatology 31 (2016) This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

2 H Miyachi et al. Management of T1 colorectal cancers favorable histologic grade, and no lymphovascular infiltration. 14,15 The European Society for Medical Oncology guidelines criteria contain the limitation of tumor budding 16 and absence of deeper invasion into the submucosa in addition. 17,18 But, in terms of deeper invasion, it is not clearly defined. Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines consist of similar criteria, except it explicitly refers to the depth of invasion as < 1000 μm. 19 Consequently, additional surgery is considered when the invasion depth is 1000 μm or more. However, the merit of this 1000-μm rule is still debated In addition, LNM occurs in around 10% of patients with surgically resected T1 carcinoma, 23 and hence, many patients without LNM undergo surgery. This unnecessary use of surgery has become a major issue. 3,24,25 Given the issues described earlier, it is clear that the indication for additional surgery is still debatable. In addition, there is little information concerning LNM-free condition. We conducted this study to clarify the risk factors for LNM in a large cohort of T1 carcinoma patients and to stratify LNM risk thus potentially reducing the excessive adaptation of surgery after endoscopic treatment. Methods Patients and clinical data. Between April 2001 and November 2014, a total of 23, 725 colorectal neoplasms excluding advanced carcinomas were resected endoscopically or surgically at our hospital. Written informed consent was obtained from all the patients before endoscopy. Of these, 1000 T1 colorectal carcinomas were included. Patients who were diagnosed with familial adenomatous polyposis (n = 1), Lynch syndrome (n = 3), ulcerative colitis (n = 1), those who underwent only endoscopic treatment (n = 302), those who underwent transanal endoscopic microsurgery (n = 4), and those who underwent surgery because of a synchronous invasive carcinoma (n = 25) were excluded. A pathological evaluation was not possible in 11 cases because of the specimen s damage or lost. Thus, for 653 cases, initial (n = 342) or additional (n = 311) surgeries with nodal dissection were performed in accordance with the principals of the JSCCR guidelines. The age and sex of the patients, the location, pit pattern, and morphology of the lesions were reviewed from the electronic record system. The pit pattern was graded according to Kudo s pit pattern classification. 7 Endoscopic morphology was classified as a depressed, flat, or protruded type according to Kudo s morphological/development classification. 26 Pathological evaluation. All resected lesions were retrieved and immediately fixed in a 10% buffered formalin solution for h and were elaborately observed with a focus on the pit pattern using a stereomicroscope. Subsequently, they were cut at the point where the deepest invasion area could be exposed on the cut end surface. And the other histological specimens were cut into parallel 2- to 3-mm-thick sections. Tumor size was measured after formalin fixation. The specimens were examined by one pathologist based on the World Health Organization (WHO) criteria 27 and the JSCCR guidelines. 19 The histological grade, tumor budding, DR, BGA, growth type, and the presence of LNM were investigated using HE-stained specimens. Lymphatic infiltration and status of the MM were evaluated using HE staining and immunostaining with anti-d2-40 and desmin antibodies (both from Dako North America Inc., Carpinteria, CA, USA). Vascular infiltration was evaluated using double staining with HE and Victoria blue (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). Histological grade was based on the least differentiated tumor component and was classified according to WHO criteria. 27 In this study, a poorly differentiated adenocarcinoma or mucinous carcinoma (Por/Muc) component was considered present if any part of the lesion contained either of these features. Tumor budding was defined as an isolated single cancer cell or a cluster of fewer than five carcinoma cells at the invasive front. 28 The field where budding was most intensive was selected to count the number of buds with a 20 objective lens. Fields with five or more buds were considered to be positive. 11 BGA was defined as an adenomatous component contiguous to carcinoma. 8 Growth type was evaluated into polypoid growth and non-polypoid growth. 29 The vertical invasion depth was measured according to the JSCCR guidelines. 19 Evaluation of the MM. The status of the MM (MM grade) was evaluated using desmin immunostaining and classified as either grade 1, when muscular fibers were still maintained, or grade 2 when the muscle fibers had fragmented or disappeared (Fig. 1). Hence, if the muscular fibers of a lesion maintained their original directionality and continuity but had disappeared only a small part (within three to four normal glands wide) because of carcinoma invasion, the status of MM was judged as grade 1. However, if there were any controversial points on these conditions, all cases fell into grade 2. Specifically, if the muscular fibers were fragmented and had lost their original alignment or showed wider disappearance, the status of the MM was regarded as grade 2. A typical case of MM grade 2 is shown in Figure 2. Validation study for MM grade evaluation. Fifty pathological images of both HE and desmin immunostaining for each lesion were randomly selected from among all the lesions for the first assessment by the two pathologists (S. H. and K. K.). Four weeks later, the same images were randomly allocated for the second assessment. Inter-observer agreement was calculated from the results of the first diagnosis. Cohen s kappa values of less than 0, , , , , and were considered to be poor, slight, fair, moderate, substantial, and almost perfect, respectively. 30 Statistical analysis. We analyzed the clinicopathological factors listed earlier using Fisher s exact test and Welch test. A P-value less than 0.05 was considered significant. Multivariate stepwise logistic regression analysis with regard to LNM was subsequently performed to calculate odds ratios and 95% confidence intervals (CIs) after accounting for potential confounders. Finally, based on significant factors, we stratified the T1 carcinoma patients according to their LNM risk. All data are presented as the mean ± standard deviation. We conducted all analyses using R version ( Journal of Gastroenterology and Hepatology 31 (2016)

3 Management of T1 colorectal cancers H Miyachi et al. Figure 1 The status of the muscularis mucosae (MM grade): (a) MM grade 1 is indicated when maintained muscular fibers are still present. (b and c) Histology of a typical lesion characterized by the maintenance of the muscularis mucosae (MM grade 1) by HE staining and desmin immunostaining. The muscular fibers maintain their original directionality and continuity but had disappeared only a small part because of carcinoma invasion. (d) MM grade 2 corresponds to a condition in which they have become fragmented or disappeared. (e and f) Histology of a typical lesion characterized by the fragmentation or disappearance of the muscularis mucosae (MM grade 2), as assessed using HE staining and desmin immunostaining. The muscular fibers were fragmented and lost their original alignment, or showed wider disappearance. Figure 2 A typical case of muscularis mucosae (MM) grade 2: (a) A 8-mm-sized lesion with erythematous color was observed in white light observation. This lesion was located in the sigmoid colon. (b) In indigocarmine-sprayed magnifying observation, the dye was accumulated in circumferentially grooved margin. Because the margin with depression was recognized, this lesion was diagnosed as Is+IIc. (c) In crystal violet-stained magnifying observation, non-structure area was seen around irregular pits, which was diagnosed as type V N pit pattern. Initial laparoscopic-assisted surgery was performed. (d and e) Histology of this lesion by HE staining and desmin immunostaining. The pathological diagnosis was T1 carcinoma (SM 3750 μm), type Is+IIc, 8 mm, well-to-moderately differentiated adenocarcinoma, ly1, v1, Budding +, pn1. The muscular fibers were fragmented and showed wider disappearance. (f) Histology of the metastatic lymph node by HE staining. Ethical considerations. This study was approved by the Institutional Review Board in our hospital ( ) and was registered in the University Hospital Medical Network Clinical Trials Registry (UMIN ). Results Clinical characteristics. Initial or additional surgery with lymph node dissection was performed for each of the 653 patients, 1128 Journal of Gastroenterology and Hepatology 31 (2016)

4 H Miyachi et al. Management of T1 colorectal cancers 408 of whom were men and 245 were women, with a mean age of 65.1 ± 11.3 years (range, years). The mean tumor size was 21.0 ± 12.4 mm (range, mm). There were 198 depressed-type, 178 flat-type, and 277 protruded-type lesions. And 463 lesions were located in the colon, and 190 were in the rectum. Comparisons of pathological factors between MM grade 1 and MM grade 2. Table 1 shows the comparisons of pathological factors and invasion depth between MM grade 1 and MM grade 2 lesions. The rates of lymphovascular infiltration, tumor budding, a Por/Muc component, and DR on the superficial layer in MM grade 2 lesions were significantly higher than those in MM grade 1. The invasion depth was significantly longer in MM grade 2 group. Relationships between clinicopathological factors and LNM. Lymph node metastasis was found in 60 (9.2%) of the 653 cases. Table 2 lists clinicopathological factors with respect to LNM. Female patients were at a significantly higher risk of LNM than male patients. Regarding pathological factors, lymphovascular infiltration, tumor budding, a Por/Muc component, and MM grade 2 were significant. In contrast with MM grade 2, no patients with MM grade 1 lesions had LNM. We therefore performed multivariate analyses on the 609 patients with MM grade 2 lesions. This revealed that attribution of female, lymphovascular infiltration, tumor budding, and a Por/Muc component remained as independent factors. The incidence of LNM based on the combination of risk factors. Table 3 shows the rates of LNM according to the combination of significant factors for female and male patients with MM grade 2 lesions. The incidence of LNM in female and male patients without any pathological factors was 1.2% (1/81) and 0.9% (1/107), respectively, which increased to 20.1% (30/149) and 10.3% (28/272) if at least one factor was present. Table 1 Comparisons of pathological factors between MM grade 1 and MM grade 2 Pathological factors No. of cases with/without pathological factor (%) OR (95% CI) P MM grade 1 (n = 44) MM grade 2 (n = 609) Lymphovascular infiltration (+/ ) 4/40 (9.1%/90.9%) 357/252 (58.6%/41.4%) 0.07 ( ) < Tumor budding (+/ ) 3/41 (6.8%/93.2%) 182/427 (29.9%/70.1%) 0.17 ( ) < Por/Muc component (+/ ) 2/42 (4.5%/95.5%) 114/495 (18.7%/81.3%) 0.21 ( ) DR on the superficial layer (+/ ) 0/44 (0.0%/100%) 204/405 (33.5%/66.5%) 0 (0 0.18) < Background adenoma (absence/presence) 25/19 (56.8%/43.2%) 405/204 (66.5%/33.5%) 0.66 ( ) Growth type (NPG/PG) 14/30 (31.8%/68.2%) 210/399 (34.5%/65.5%) 0.89 ( ) 0.87 Depth of invasion (mean ± SD) (μm) ± ± N/A < CI, confidence interval; DR, desmoplastic reaction; MM grade, status of the muscularis mucosae; Muc, mucinous carcinoma; N/A, not applicable; NPG, non-polypoid growth; No, number; OR, odds ratio; Por, poorly differentiated adenocarcinoma; PG, polypoid growth; SD, standard deviation. Table 2 Relationships between clinicopathological factors and LNM Clinicopathological factors No. of cases with LNM (n = 60) No. of cases without LNM (n = 593) Univariate analysis Multivariate analysis OR (95% CI) P OR (95% CI) P Age ( 70/< 70 years) 21/39 (35.0%/65.0%) 230/363 (38.8%/61.2%) 0.85 ( ) Sex (female/male) 31/29 (51.7%/48.3%) 214/379 (36.1%/63.9%) 1.89 ( ) ( ) Tumor size ( 20/< 20 mm) 32/28 (53.3%/46.7%) 288/305 (48.6%/51.4%) 1.21 ( ) Location (rectum/colon) 21/39 (35.0%/65.0%) 169/424 (28.5%/71.5%) 1.35 ( ) Morphology (depressed/non-depressed) 17/43 (28.3%/71.7%) 181/412 (30.5%/69.5%) 0.90 ( ) Pit pattern (V N,V I high-grade/others) 43/16 (72.9%/27.1%) 426/162 (72.4%/27.6%) 1.02 ( ) Initial surgery/additional surgery 26/34 (43.3%/56.7%) 316/277 (53.3%/46.7%) 0.67 ( ) Depth of invasion ( 1000/< 1000 μm) 55/5 (91.7%/8.3%) 537/56 (90.6%/9.4%) 1.15 ( ) Lymphovascular infiltration (+/ ) 56/4 (93.3%/6.7%) 305/288 (51.4%/48.6%) ( ) < ( ) <0.001 Tumor budding (+/ ) 33/27 (55.0%/45.0%) 152/441 (25.6%/74.4%) 3.54 ( ) < ( ) Por/Muc component (+/ ) 21/39 (35.0%/65.0%) 95/498 (16.0%/84.0%) 2.82 ( ) ( ) DR on the superficial layer (+/ ) 22/38 (36.7%/63.3%) 182/411 (30.7%/69.3%) 1.31 ( ) Background adenoma (absence/presence) 36/24 (60.0%/40.0%) 394/199 (66.4%/33.6%) 0.76 ( ) Growth type (NPG/PG) 17/43 (28.3%/71.7%) 207/386 (34.9%/65.1%) 0.74 ( ) MM grade (grade2/grade1) 60/0 (100.0%/0.0%) 549/44 (92.6%/7.4%) Inf Analysis among the 609 patients with MM grade 2 lesions. Excluding six cases in which the pit pattern was not ascertained. CI, confidence interval; DR, desmoplastic reaction; Inf, infinity; LNM, lymph node metastasis; MM grade, status of the muscularis mucosae; Muc, mucinous carcinoma; NPG, non-polypoid growth; No, number; OR, odds ratio; PG, polypoid growth; Por, poorly differentiated adenocarcinoma. Journal of Gastroenterology and Hepatology 31 (2016)

5 Management of T1 colorectal cancers H Miyachi et al. Table 3 Incidence of LNM according to the combination of the significant pathological factors among the MM grade 2 cases Combination of factors Reliability of MM grade evaluation. There was substantial inter-observer agreement with a kappa value of 0.67 (95% CI, ). Perfect and substantial intra-observer agreements were obtained with a kappa value of 1.0 (95% CI, 1) for S. H. and 0.71 (95% CI, ) for K. K. Discussion Female No. of cases with LNM (%) Total no. of cases Male No. of cases with LNM (%) Total no. of cases None 1 (1.2%) 81 1 (0.9%) 107 Ly/v 10 (18.5%) 54 6 (5.1%) 118 Bu 0 (0.0%) 8 0 (0.0%) 18 Por/Muc 1 (7.7%) 13 0 (0.0%) 20 Ly/v, Bu 10 (21.7%) (17.5%) 63 Ly/v, Por/Muc 2 (18.2%) 11 6 (26.1%) 23 Bu, Por/Muc 0 (0.0%) 3 1 (50.0%) 2 Ly/v, Bu, Por/Muc 7 (50.0%) 14 4 (14.3%) 28 Total 31 (13.5%) (7.65%) 379 Bu, tumor budding; LNM, lymph node metastasis; Ly/v, lymphovascular infiltration; No, number; Por/Muc, poorly differentiated adenocarcinoma or mucinous carcinoma. In this study, the incidence of pathological factors was significantly lower in MM grade 1 lesions, and no patients with an MM grade 1 lesion had LNM, indicating that MM grade 1 was a favorable predictive factor in this respect. These patients could be categorized as being in an ultralow-risk group. Among the Figure 3 The risk stratification of LNM among patients with T1 colorectal carcinoma: the incidence of LNM (95% CI) is given for each group. CI, confidence interval; LNM, lymph node metastasis; Muc, mucinous carcinoma; Por, poorly differentiated adenocarcinoma. patients with MM grade 2 lesions, those with T1 carcinomas without lymphovascular infiltration, tumor budding, or a Por/Muc component may only require monitoring, as their risk of LNM is about 1%. These could be categorized into a low-risk group. Patient sex was a further risk factor in the other cases, as there was an incidence of LNM of around 10% in male patients (categorized in a high-risk group ) and about 20% in female patients (categorized as an ultrahigh-risk group ) (Fig. 3). These patients should need additional surgery with lymph node dissection. When we applied this risk stratification of LNM to the 653 patients who had undergone surgery in this study, we found that 196 of them may not have actually required surgery, and indeed, none of these patients had LNM. Moreover, 189 of these 196 cases had no pathological factors but showed an invasion depth of 1000 μm, which means that 189 unnecessary surgeries might have been performed merely because of the 1000-μm rule. Similarly, expanding the criteria for curative endoscopic resection has been considered in a few papers. 24,25 These studies found that the incidence of LNM was less than 2% regardless of the invasion depth, if the lesions showed favorable histology, negative lymphovascular infiltration, and limited tumor budding. In terms of reducing the number of unnecessary additional surgeries, this stratification using MM grade better reflected the risk of LNM than the current criteria using the invasion depth. Kitajima et al. reported that the rate of LNM was 0% if the depth was < 1000 μm. 31 Another review showed the depth of 1000 μm was a risk factor of LNM. 32 In this study, however, the invasion depth turned out not to be significant, because five lesions developed LNM among 61 cases with < 1000 μm. It does not mean that this result lacks in consistency with, for example, the following cases. According to Suh et al., 8 12 out of 98 sm1 (< 1000 μm or infiltration into the upper third) lesions had LNM. Likewise, one case by Ueno et al., 11 one case by Tateishi et al., 12 two cases by Nakadoi et al., 25 four cases by Choi et al., 33 and one case by Yoshida et al. 34 Table 4 shows the five cases in our facility. Of these, three cases had any of the pathological factors. It might be sufficiently considered that some lesion with < 1000 μm had a pathological factor and would develop LNM. The other two cases had LNM without pathological factors. Because these cases showed MM grade 2, they were just corresponded to low-risk group in this risk stratification. At least we could say that the rate of LNM in low-risk group was 1.06% (Fig. 3). Such cases were also reported. 8,34 As another point of view, the method of measuring an invasion depth affects the incidence of cases with < 1000 μm. The differences of measured depth might occur depending on the pathologists, even though such bias falls into an inevitable limitation. Moreover, several problems about measuring the depth and 1000 μm border were pointed out In detail, we discussed these issues in another paper. 35 The policy of considering not only the vertical depth but also horizontal extent of invasion even within superficial submucosa has been incorporated into to the MM grade. We used Kudo s classification of the degree of submucosal invasion, 36 which has three categories of vertical depth: sm1, sm2, and sm3. Importantly, sm1 is further classified based on the horizontal extension within the superficial submucosa. Even when a lesion shows shallow invasion, the invasion spread widely in the horizontal direction is categorized as sm1c, which falls into massively invasive Journal of Gastroenterology and Hepatology 31 (2016)

6 H Miyachi et al. Management of T1 colorectal cancers Table 4 Detailed data for the cases with < 1000 μm having LNM Case no. Age Sex Size (mm) Location Morphology Depth (μm) MM Ly/v Bu Por/Muc DR BGA GT 1 54 M 13 Sigmod Protruded PG 2 61 F 20 Sigmod Protruded PG 3 57 M 30 Sigmod Protruded PG 4 54 M 35 Rb Flat PG 5 50 M 7 Sigmod Protruded PG BGA, background adenoma; Bu, tumor budding; Depth, depth of invasion; DR, desmoplastic reaction on the superficial layer; F, female; GT, growth type; LNM, lymph node metastasis; Ly/v, lymphovascular infiltration; M, male; MM, MM grade (status of the muscularis mucosae); No, number; PG, polypoid growth; Por/Muc, poorly differentiated adenocarcinoma or mucinous carcinoma. Likewise, focusing on the horizontal extent or the status of the MM, Okabe et al. (1998) used both the depth and width of invasion as a major prognostic indicator of T1 carcinomas. 9 In 2004, they also demonstrated that the invasion width was a predictor of LNM among 304 carcinomas, although it was not significant. 10 Suzuki et al. 4 measured the depth, width, and area of invasion in 65 lesions and found a significant relationship between LNM and the width but not the depth or the area. Tateishi et al. 12 tried to select patients with LNM without measuring the depth and proved that a completely disrupted MM was a significant risk factor in 322 lesions. Nakadoi et al. classified the status of the MM into three groups, possible to identify or estimate, not possible to identify or estimate, and complete disappearance, and found that the latter was an independent factor among 276 patients. 6 In contrast, we could not find that MM with a complete disappearance status was significant. Among our 609 cases with MM grade 2, the rate of LNM in tumors showing complete disappearance was 10.2% (28/274), while that of lesions with incomplete disappearance was 9.6% (32/335) (P = 0.79). This is one of the reasons why we did not distinguish between incomplete and complete disappearance within MM grade 2. An additional advantage of using the MM grade is that it could potentially be used to exclude the possibility of LNM. Saraste et al. 37 also performed a risk stratification for LNM among 205 T1 rectal carcinoma patients, but they could not identify which patients would not require additional surgery. There have only been a few reports of factors that accurately predict LNM-free survival. Ueno et al. 11 found that LNM was never observed in patients with the lesions that invaded less than 2000 μm in width or 500 μm in depth. Tominaga et al. 13 reported that patients with a lesion showing a well-preserved MM had no LNM. We also found that patients with MM grade 1 formed an ultralow-risk group. Its 95% CIs for the rate of LNM was wide, although this was probably due to the small number of MM grade 1 lesions. It is possible, of course, that a patient with a MM grade 1 lesion could develop LNM, but this is likely to be a rare event. This is supported by the meta-analysis performed by Mou et al., 23 in which patients with well-differentiated T1 carcinomas without lymphovascular infiltration or tumor budding were found to have the lowest risk of LNM (1.9%; 95% CI, %), but the risk was not zero. We found that female patients were at a higher risk of LNM than male patients, if they had MM grade 2 lesions together with any significant pathological factors. However, there were no significant differences between male and female patients with respect to age, location, morphology, or the incidence of pathological factors. There are several reports describing the association between patient sex and LNM. Of these, a few showed a tendency for female patients to develop LNM more frequently, but these differences were not significant. 6,31 No previous reports have definitively revealed a significant relationship between patient sex and LNM risk in patients with T1 carcinomas, although its basis is unclear, and further research will be needed. There are some potential limitations. First, this was a singleinstitutional investigation; consequently, there could be a selection bias. However, the pathological specimens were examined in the same manner, 12 and the sample size was larger than any other single-institutional reports published so far. Second, MM grade was judged by one pathologist; therefore, some diagnostic bias could exist. Nevertheless, MM grade is a simple classification and easy to assess, and the use of desmin immunostaining could improve the diagnostic objectivity. Indeed, good observer agreements were confirmed. Third, all the study patients underwent surgical resection in order to investigate the presence of LNM. The risk factors identified here are not necessarily applicable to patients treated by endoscopic resection alone. Further investigation into recurrence will be needed. To conclude, MM grade is a simple and reproducible factor for predicting LNM based on the width of submucosal invasion. Stratification using the MM grade, significant pathological factors, and patient sex offered a more appropriate indication for additional surgery without measuring the invasion depth among patients with T1 colorectal carcinoma. In addition, this stratification can predict which patients will remain LNM-free and could help to reduce unnecessary surgery. Stated simply, for cases with MM grade 2 and with lymphovascular infiltration, tumor budding, and/or a Por/Muc component, additional surgery with lymph node dissection should be recommended, especially for female patients. Acknowledgments The authors express great thanks to Chiaki Nishimura for advising on statistical analyses, to Kenichi Matsui and Osamu Henmi for helping with the statistical analyses, to Naomi Takenaka for creating the illustrations, to Yoko Tanaka for the assistance in English composition, and to all members of the Digestive Disease Center and the Department of Pathology, Showa University Northern Yokohama Hospital for their excellent assistance. Journal of Gastroenterology and Hepatology 31 (2016)

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