Desmoplastic Reaction in Biopsy Specimens of T1 Stage Colorectal Cancer Plays a Critical Role in Defining the Level of Sub-Mucosal Invasion

Early CRC is defined as a tumour whose invasion is limited to the mucosa or submucosa. The endoscopic mucosal resection and endoscopic mucosal dissection (EMR/ESD) have be‐ come useful for early CRC. However, EMR/ESD is applicable only to intramucosal carcino‐ ma, and additional surgery is required if the resected lesion reveals submucosal invasion by pathological diagnosis. Therefore, endoscopist and surgical pathologist considered that it would be very useful to predict the depth of invasion of submucosal invasive CRC before EMR/ESD. On the other hand, desmoplastic reaction (DR) which is characterized by the infiltration of eosinophilic myofibroblasts in the stroma of invasive carcinoma is suggested to be a prognostic marker in CRC patients.


Introduction
Observation of the mucosal crypt patterns using chromoendoscopy with magnification has been reported to be the most reliable method for determining whether a colorectal cancer (CRC) is early or advanced tumour.Moreover, brand-new endoscopic system have the capability to enhance visibility by the capillary pattern, which may prove to be reliable for detection of deep submucosal invasive CRC.
Early CRC is defined as a tumour whose invasion is limited to the mucosa or submucosa.The endoscopic mucosal resection and endoscopic mucosal dissection (EMR/ESD) have become useful for early CRC.However, EMR/ESD is applicable only to intramucosal carcinoma, and additional surgery is required if the resected lesion reveals submucosal invasion by pathological diagnosis.Therefore, endoscopist and surgical pathologist considered that it would be very useful to predict the depth of invasion of submucosal invasive CRC before EMR/ESD.On the other hand, desmoplastic reaction (DR) which is characterized by the infiltration of eosinophilic myofibroblasts in the stroma of invasive carcinoma is suggested to be a prognostic marker in CRC patients.
Here we describe that detection of DR in pre-treatment biopsy specimens is useful for predicting the depth of submucosal invasion and evaluation of submucosal depth with head invasion or stalk invasion in post EMR/ESD specimen is useful for predicting the lymph node metastasis and discuss relevant issues in arriving at the correct differential diagnosis based on histological findings for gastrointestinal endoscopist and surgical pathologist.

Correlations between lymph node metastasis and early colorectal carcinoma
The endoscopic mucosal resection and endoscopic submucosal dissection (EMR/ESD) have become useful for early CRC which is defined as a T1 stage tumour whose invasion is limited to the mucosa or submucosa according to the T categories for colorectal cancer of the American Joint Committee on Cancer (AJCC) staging system (Table 1).
Tx: No description of the tumor's extent is possible because of incomplete information.
Tis: The cancer is in the earliest stage (in situ).It involves only the mucosa.It has not grown beyond the muscularis mucosa (inner muscle layer).T1: The cancer has grown through the muscularis mucosa and extends into the submucosa.T2: The cancer has grown through the submucosa and extends into the muscularis propria (thick outer muscle layer).
T3: The cancer has grown through the muscularis propria and into the outermost layers of the colon or rectum but not through them.It has not reached any nearby organs or tissues.
T4a: The cancer has grown through the serosa (also known as the visceral peritoneum), the outermost lining of the intestines.
T4b:The cancer has grown through the wall of the colon or rectum and is attached to or invades into nearby tissues or organs.
Table 1.T categories for colorectal cancer of the AJCC staging system.
It has been known that we should be considered be additional resection is required due to the risk of lymph node metastasis following findings (1) massive submucosal invasion: (2) lymphatic/vessel invasion; or (3) poorly differentiated component in resected EMR/ESD specimens [7,8].There has been no standard method of measurement of submucosal invasion depth.Therefore, Japanese Society for Cancer of the Colon and Rectum has recently demonstrated definite for method of measurement of submucosal invasion depth.

Ip type (pedunculated lesion) and non-Ip type (nonpedunculated lesion) of early colorectal carcinoma
Macroscopic type (endoscopic finding) was assessed according to the macroscopic classification of early stomach carcinoma, with minor modifications.In shortly, SICCs were divided into two lesions: pedunculated (Ip type) (Figure.The method used for measurement of submucosal invasion depth (Figure .3) Firstly, EMR/ESD resected specimens are divided into pedunculated(Ip type) and nonpedunculated(Non-Ip type).For pedunculated SICC, level 2 according to Haggitt's classification [9] was used as the baseline (so-called Haggitt's line), and submucosal invasion depth was measured as the vertical distance from this line to the deepest site of invasion.The baseline to distinguish between head invasion, (Figure.).In head invasion, submucosal invasion depth was regarded as 0 µm.When the deepest portion of invasion was located below the baseline, the case was defined as a stalk invasion and the vertical distance from this line to the deepest portion of invasion was utilized as submucosal invasion depth.
On the other hand, nonpedunculated and when the muscularis mucosae could be identified in hematoxylin and eosin stain, the muscularis mucosae was used as baseline and the vertical distance from this line to the deepest site of invasion represented submucosal invasion depth, (Figure .4(c))., however, when the muscularis mucosae could not be identified, the superficial aspect of the SICC was used as baseline, (Figure.4(d)).

Correlations between lymph node metastasis and depth of submucosal invasive colorectal carcinoma
The Japanese collaborative retrospectively study for 865 SICCs.This nationwide survey not only represents a first for Japan, but reviewing the literature using PubMed revealed no similar surveys from anywhere in the world at that time [3] This study reported that pedunculated (Ip type) SICC, rate of lymph node metastasis was never in head invasion cases and stalk invasion cases with submucosal invasion depth < 3000 µm if lymphatic invasion was negative (Table 2).
And, For nonpedunculated (non-Ip type) SICC, rate of lymph node metastasis was also 0% if submucosal invasion depth was <1000 µm (

The treatment of colorectal carcinoma with submucosal invasion state after endoscopic resection
Many new treatment methods have been developed over the last few decades.The Japanese Society for Cancer of the Colon and Rectum guidelines 2010 for the treatment of colorectal cancer (JSCCR Guidelines 2010 [13]) have been prepared to show standard treatment strategies for submucosal invasive colorectal cancer (Figure . 6).

Detection of desmoplastic reaction in biopsy specimens of colorectal cancer
Early CRC, including submucosal invasive colorectal carcinoma (SICC), is defined as a tumor whose invasion is limited to the mucosa or submucosa.The endoscopic resection (EMR/ESD) have been useful for early CRC.However, endoscopic resection is applicable only to intramucosal carcinoma (Tis stage tumor), and additional surgery is required if the resected specimen reveals submucosal invasion by pathological diagnosis.However, the study of Japanese Society for Cancer of the Colon and Recum have recently demonstrated that the depth of submucosal invasion is closely correlated with the prevalence of lymph node metastasis in patients with SICC (See.Correlations between lymph node metastasis and early colorectal Carcinoma).
Therefore, gastrointestinal endoscopist and surgical pathologist considered that it would be very useful to predict the depth of invasion of SICC before endoscopic resection for case selection.New endoscopic systems are possible to predict submocosal invasion depth without biopsy.However, these developed systems have not been used in everywhere.On the other hand, the stromal change associated with carcinoma invasion has been called desmoplastic reaction (DR), desmoplasia, and cancer-associated fibroblast.Reported incidence of the DR is suggested to be a prognostic marker in CRC patients [16,17].Recently, JSCCR studies have been assessed the DR in pretreatment biopsy specimens of SICRC to predict the submucosal depth in retorospective and prospective study.
The DR is characterized by modifications in the composition of stromal cells and extracellular matrix (ECM) components with eosinophilic change [18,19] (Figure . 8).The main producers of many ECM compounds and represent the major cellular component including DR that often show differentiated phenotype with expression of the smooth muscle actin, platelet derived growth factor receptor-type I collagen [18,20,21].
The presence and histological findings of DR in biopsy specimens were evaluated by pathologists at each respective institute and the criteria for assessment of DR established in consensus meeting among JSCCR members including us.(Table 6). 1) Existence of carcinoma is required for detection of DR.
2) The histological findings of infiltrating carcinoma do not signify the presence of DR.
3) DR contains areas of collagen fiber accumulation and myofibroblast proliferation, but inflammatory infiltration does not signify the presence of DR.
4) All histological findings were determined by HE stain alone; detection of DR does notrequire the use of any special stains.
DR, desmoplastic reaction.Table 6.Criteria for detection of desmoplastic reaction (adapted from [22]) A retorospective study for detection of desmoplastic reaction in biopsy specimens of early colorectal cancer reported that 359 patients with SICRCs, who had undergone surgical or endoscopic mucosal resection, were analysed [23].For pedunculated (Ip type) SICRCs was not significantly related to submucosal depth.However, for nonpedunculated (non-Ip type) the prevalence of DR in pretreatment biopsy specimens was significantly related to submucosal depth.When nonpedunculated(non-Ip type) SICRCs were further divided using a specific cut off value of 1000 µm for submucosal depth, the positivity ratio of DR in pretreatment biopsy specimens was significantly higher in SICRCs with an submucosal depth of 1000 µm than in cases where the submucosal depth was <1000 µm (Table 7).Detection of DR in pretreatment biopsy specimens is useful for the prediction of submucosal depth in nonpedunculated (non-Ip) SICRCs, After retorospective study [22], same study group confirmed verification of patients SICRC with 112 nonpedunculated (non-Ip type) cases.Finally, nonpedunculated (non-Ip type) case of the prevalence of DR was significantly correlated with submucosal depth.The sensitivity and specificity of detection of DR for prediction of pSM2 (tumor invasion <1000 µm) in nonpedunculated SICRC were 68.6% and 92.0%, respectively.
Additionally, receiver operating characteristic (ROC), analysis confirmed 950 µm as the best diagnostic cut-off value of submucosal depth for DR detection, and 50 µm, which is the difference between the value of 950 µm as determined by cut off value (COV) and 1000 µm, the defining value of pSM2, is an acceptable measurement error range.
In statistics and diagnostic testing, positive predictive value (PPV) is the proportion of patients with positive test results who are correctly diagnosed, on the other hand, negative predictive value (NPV) is negative test results.Both of PPV and NPV are critical measure of the performance of a diagnostic method.In this studies revealed that PPV:0.93,NPV:0.59 in pedunculated and PPV:0.95,NPV:0.59 in nonpedunculated type.These results provide a basis for assessment of DR as a good indicator of pSM2.(Table 8).

Conclusion
In this issue, we have discussed the a critical role of pathological assessment for T1 stage colorectal cancer, several problems related to the pathological diagnosis of early CRC at increased risk of lymph node metastasis and submucosal invasion.A new endoscopic systems which may prove to be reliable for detection of deep submucosal invasive CRC.Moreover, current endoscopic resection (EMR/ESD) have become useful for early CRC, but, these resection is applicable only to intramucosal carcinoma, and additional surgery is required if the resected lesion reveals submucosal invasion by pathological diagnosis, because prevalence rates of lymph node metastasis about 10% of all patients with submucosal invasive colorectal carcinoma.
We believe that curative endoscopic management for early CRC may be need to accurately pathological diagnosis of submucosal depth.Assessment of DR in pretreatment biopsy specimen nonpedunculated (non-Ip type) and submucosal depth with head invasion or stalk invasion in pedunculated (Ip type) for post endoscopic resection (EMR/ESD) specimen may be useful for the clinicopathological diagnosis of colorectal carcinoma with invasion into the submucosal layer.

Figure 3 .
Figure 3. Algorithm of measurement of submucosal invasion depth.

Figure 4 .
Figure 4. Schema of measurement of submucosal invasion depth.Ip type with head invasion (a) and stalk invasion (b), non-Ip type with musucular mucosae (c) and without musucular mucosae (d).

Figure 6 .
Figure 6.Guide line for the treatment of colorectal carcinoma with submucosal invasion state after endoscopic resection.(adapted from JSCCR Guidelines 2010[13] with minor modifications );; Pap: papillary adenocarcinoma, Tub: tubular adenocarcinoma, Por: poorly differentiated adenocarcinoma, Sig: signet-ring cell carcinoma, Muc: mucinous adenocarcinoma, SM: submucosal invasion, Budding: tumor budding.The tumor budding denotes that at the invasion front of colorectal adenocarcinomas tumour cells, and the potential of tumour budding as a prognostic factor (G1:Grade 1 to G3: Grade 3) for routine surgical pathology[14,15])

Figure 7 .
Figure 7. Endoscopic, stereomicroscopic, macroscopic findings of the early colorectal carcinoma.Endoscopic finding of the pedunculated(Non-Ip) type (a), several pits are arranged irregularly in the stereomicroscopic view (b), macroscopic appearance of SICC, post formalin-fixed.Most of the endoscopic detectable lesions were the irregular elevated type macroscopically.(White bar: mucosal invasion, Red bar: submucosal invasion) (c),

Figure 8 .
Figure 8. Histological appearance of desmoplastic reaction (DR).Note the growth of eosinophilic spindle cells (myofibroblasts.*)with submucosal invasive carcinoma component on Hematoxylin and Eosin staining section.

Table 3 )
[9,10]ultivariate analysis, SM depth <1000µm (P <0.006), sprouting (P <0.002), and lymphatic invasion (P<0.0001)representedsignificantrisk factors, with odds ratios of 5.404, 2.276, and 4.691, respectively.Several priviously reports suggested that prognosis in patients with early colorectal carcinoma based on Haggitt's classification, finding that level 4 which is carcinoma invading the submucosa of the bowel wall below the stalk of the polyp but above the muscularis propria, represented the most important factor for lymph node metastasis[9,10].Therefore, these results revealed that submucosal invasion could be an important of predicting lymph node metastasis potential.

Table 2 .
[3]ationship between clinicopathological factors and the rate of lymph node metastasis according to SM depth in pedunculated (Ip type) SICC (adapted from[3])

Table 3 .
[3]ationship between clinicopathological factors and the rate of lymph node metastasis according to SM depth in nonpedunculated (non-Ip type) SICC (adapted from[3])

Table 4 .
[11]tionship between clinicopathological factors and the rate of the lymph node metastasis according to SM depth (summarized of Table2 and 3, adapted from[11].English translated version]

Table 7 .
[23]tionship between depth of submucosal invasion and DR in biopsy specimens of patients with SICRCsretorospective study -(adapted from[23])

Table 8 .
[22]tionship between depth of submucosal invasion and DR in biopsy specimens of patients with pedunculated (upper part) and nonpedunculated (lower part) type -prospective study -(adapted from[22])