In 2010, the World Health Organization categorized L-cell type neuroendocrine tumors (NETs) as tumors of uncertain malignancy, while all others were classified as malignant. However, the diagnostic necessity of L-cell immunophenotyping is unclear, as are tumor stage and grade that may guide diagnosis and management. To clarify the predictive markers of rectal neuroendocrine neoplasms (NENs), 5- and 10-year overall survival (OS) was analyzed by pathological parameters including L-cell phenotype.
A total of 2,385 rectal NENs were analyzed from our previous multicenter study and a subset of 170 rectal NENs was immunophenotyped.
In univariate survival analysis, tumor grade (p < 0.0001), extent (p < 0.0001), size (p < 0.0001), lymph node metastasis (p=0.0063), and L-cell phenotype (p < 0.0001) showed significant correlation with the prognosis of rectal NENs; however, none of these markers achieved independent significance in multivariate analysis. The 10-year OS of tumors of NET grade 1, < 10 mm, the mucosa/submucosa was 97.58%, 99.47%, and 99.03%, respectively. L-Cell marker, glucagon II (GLP-1&2), with a cut off score of > 10, is useful in defining L-Cell type. In this study, an L-cell immunophenotype was found in 83.5% of all rectal NENs and most, but not all L-cell type tumors were NET G1, small (< 10 mm) and confined to the mucosa/submucosa.
From these results, the biological behavior of rectal NENs does not appear to be determined by L-cell type alone but instead by a combination of pathological parameters.
A remarkably increasing incidence of gastroentero-pancreatic neuroendocrine neoplasms has been reported over the past several decades throughout the world [
The WHO updated the classification of these tumors in 2010 and categorized them all as malignant, except for L-cell type and tubular NETs [
In our previously published large nationwide multicenter study, rectal NENs showed the best prognosis among all gastroenteropancreatic (GEP)-NENs. However, it was unclear whether or not cell of origin affected the prognosis because information on cell type was unavailable in that study.
Here questions arise about the necessity of L-cell type immunophenotyping in daily practice for better prediction of patient’s prognosis. In addition, it is unclear whether small rectal (WHO G1) NETs by the 2010 WHO classification should be categorized as malignant, if not of L-cell type. To investigate these uncertainties, we analyzed the prognostic significance of L-cell phenotype in rectal NETs, along with previously known factors affecting biologic behavior, to assess the validity of malignancy criteria of rectal NENs.
Rectal NENs from the nationwide Korean multicenter data of our previous publication were studied [
Inclusion criteria required pathologically confirmed rectal NENs from January 1, 2000, to December 31, 2009. Exclusion criteria included adenocarcinomas with focal neuroendocrine differentiation. If a NEN underwent subsequent excision or surgical resection following an initial biopsy, the biopsy report from the same case was excluded in order to eliminate duplication of cases. Pathologic parameters analyzed included tumor size, grade, extent, lymph node metastasis, and patient survival.Tumor grading was based on mitotic count or Ki-67 labeling index as NET G1 or NET G2 recommended by the 2010 WHO classification [
A total of 170 rectal NENs with available paraffin blocks and follow-up information were selected from among the nationwide data due to the limitation of the resource. To assess the relation of L-cell phenotype with other pathologic parameters and biological behavior, we included as many cases treated by surgical resection with lymph node dissection as possible, regardless of tumor grade and added cases of NET G1 treated by local excision. For comparison, 50 gastric NENs, 32 small intestinal NENs, and 22 colonic NENs were also evaluated. An auto-stainer (Benchmark XT, Ventana Medical Systems, Roche, Tucson, AZ) minimized technical and sampling error. The UltraView Universal DAB Detection Kit was used for immunohistochemical (IHC) staining. Three different primary antibodies were used for L-cell immunophenotyping: glucagon I (GLP-1, Roche), glucagon II (GLP-1&2, LifeSpan Bioscience, Seattle, WA), and PYY (Sigma, St. Louis, MO). The steps are briefly described as follows. Paraffin-embedded tissue sections from well representative blocks were deparaffinized with xylene and rehydrated through graded alcohol solutions. Antigen retrieval consisted of warming the slide to 75°C (4 minutes), and application of cell conditioning solution No. 1 (60 minutes). Endogenous peroxidase was blocked by applying UV inhibitor (4 minutes). After washing with reaction buffer, primary antibody was applied (2 hours) in a autostainer. Slides were then rinsed with reaction buffer and added one drop each of HRP UNIV MULT, DAB, and DAB H2O2 (Ventana Medical Systems, Roche) (8 minutes each). Slides were then treated with one drop of COPPER (4 minutes) before counterstaining with hematoxylin (4 minutes). Interpretation of IHC staining for glucagon I, glucagon II, and PYY was separately evaluated based on the staining intensity (1, weaker than normal control; 2, strong as control) and distribution (% of positive cells). And then they were multiplied for an immunoscore (0-200), that was grouped as 0 (< 10), 1 (10-50), 2 (51-100), or 3 (> 100). The results were interpreted as negative (score group 0) or positive (score groups 1-3). The immunostaining results were evaluated independently by two pathologists blinded to the patients’ clinical and pathologic information. L-Cell type was determined if the sample was immunohistochemically positive (immunoscore > 10) for one or more markers [
Data were presented as numbers (%) for categorical variables. In survival analysis, we excluded cases that only underwent small biopsy. To estimate the association between eligible variables and mean overall survival time, the Kaplan-Meier test was applied together with the log-rank test for comparison of various groups. The 5- and 10-year overall survival rates in each parameter were evaluated. A Cox proportional hazard regression was performed adjusting for gender and age to determine the independent prognostic factor and the significance was determined at 95% confidence level. PASW ver. 18.0 (SPSS Inc., Chicago, IL) was used for statistical analyses. A p-value of < 0.05 was considered statistically significan.
Among 4,951 GEP-NENs in our previous nationwide data of Korea, there were 2,385 rectal NENs (48.17%) [
In survival analysis, mean survival time according to the tumor grade was 74.01±0.27 months for G1, 43.14±1.62 months for G2, and 24.85±2.28 months for G3 (p < 0.0001). The tumor extent showed prognostic significance (p < 0.0001) and mean survival time was the longest in tumors confined to the mucosa and submucosa (66.05±0.15 months) and shortest in tumors with adjacent organ invasion (22.08±3.60 months) while that in tumors with distant metastasis was 35.07±2.55 months (p < 0.0001). The size of tumor was significantly correlated with patient’s survival and the size group of 10-14 mm showed the longest mean survival time (64.90±0.66 months) in contrast to the shortest in the size group of 15-20 mm (26.08±0.57) (p < 0.0001). Mean survival time according to lymph node metastasis was 33.66±1.81 months for the node positive group, in contrast to 71.35±2.90 months for the negative group (p=0.0063). Five- and 10-year OS according to the pathological parameters are shown in
Lymph node metastasis showed significant correlation with tumor grade (p=0.0069), extent (p=0.0273), and size (p=0.0039). Among 774 NET showing G1 and < 10 mm, eight cases were confirmed as having lymph node metastasis. Although the lymph node negative group showed significantly better survival compared to node positive patients when all samples were analyzed (p < 0.0063), there was no significant survival difference between them if cases of NET G1 and G2 were selectively analyzed (mean survival time±standard deviation: 40.49±2.48 months for positive group and 74.6 months for negative group, p=0.4830) (
Three L-cell markers (glucagon I, glucagon II, and PYY) clearly demonstrated L-cells scattered in the normal mucosa which served as an internal control (
Results of clinicopathologic analysis in relation to L-cell phenotype are shown in
Among all NENs in the body, rectal NETs are known to have the best prognosis, with a 5-year survival rate of 88.3% for all stages [
In this study, we analyzed the 5- and 10-year OS of rectal NETs in relation to the pathological parameters, including L-cell typing. A full of 80.61% (n=1,151) of rectal NENs were less than 10 mm and these patients had an excellent prognosis with 10-year OS of more than 99%, if the tumor was NET G1 and confined to the mucosa-submucosa (small NET). Although univariate analysis showed significant prognostic significance of tumor grade, size, extent, lymph node metastasis and L-cell type, no independent prognostic factors were identified among them in multivariate analysis. The Colonoscopy Study Group of the Korean Society of Coloproctology reported lymph node metastasis in 5.1% (21 out of 414 rectal NETs) and was related to tumor size [
In this study, to define the L-cell phenotype by immunohistochemistry, we compared three primary antibodies (2 glucagons and 1 PYY). The glucagon I primary antibody can detect the GLP-1 but the glucagon II can detect both GLP- 1 and 2. Gastric and duodenal NENs were almost all negative for all L-cell markers (except for 3 duodenal NENs), while 33.33% of non-rectal, colonic NENs and 83.7% of rectal NENs were defined as L-cell type. Even though the immunoscores of the three markers were significantly correlated with each other and all were prognostically significant by univariate analysis (p=0.0018, p < 0.0001, and p=0.0209, respectively), the patterns of expression were heterogeneous across cases (
Although the univariate analysis suggested size, grade, extent, lymph node metastasis, and L-cell phenotype as significant prognostic factors, multivariate analysis failed to show that only a single factor may not adequately predict prognosis. This also validates the conclusion that the suggested combined criteria are suitable for assessment of the biological behavior of rectal NENs. Therefore, in practice, we recommend perform IHC staining for L-cell markers for phenotyping of rectal NET and L-cell type best define classification as uncertain malignancy or low malignant potential (ICD-O code /1) [
We reconfirm the excellent prognosis for patients with rectal NET G1 if the tumor size was less than 10 mm and confined to the mucosa/submucosa, irrespective of lymph node metastasis. L-Cell type made up 96.56% of NET G1, 96.7% of mucosa/submucosa tumors, and 92.1% of tumors < 1 cm. These findings may support that 2010 WHO classification describes L-cell type as uncertain malignancy/low malignant potential (ICD-O code /1). Because multivariate analysis failed to show that only a single factor can adequately predict patient’s prognosis, we suggest combined criteria are suitable for assessment of the biological behavior of rectal NENs.
Conflict of interest relevant to this article was not reported.
We acknowledge and thank Dr. Mary Bronner, Professor and Division Chief of Anatomic Pathology at the University of Utah for her helpful review of our manuscript. Professor Myeong-Cherl Kook at the National Cancer Center of Korea kindly provided the gastric NENs samples as a control.
Five- and 10-year overall survival rates in patients with rectal neuroendocrine neoplasms (NENs) according to the grade (A), size group (B), lymph node (LN) metastasis (C), extent (D) and L-cell type (E). Rectal neuroendocrine tumors (NETs) with < 2 mitoses per 10 high-power field’s (World Health Organization G1), confined to the mucosa-submucosa, with no lymph node metastases, and of L-cell type showed excellent prognosis. In particular, 10-year survival rate of NETs G1 confined to mucosa-submucosa was more than 99%.
Survival analysis according to lymph node metastasis (LN_Mets) in all samples (A), and neuroendocrine tumors (NET) G1 and G2 (B). LN_Mets was significantly related with poor prognosis in all rectal neuroendocrine neoplasms examined (p < 0.0001), but it was not if NET G1 and G2 tumors were selectively analyzed (p=0.4830).
Patterns and distribution of immunohistochemical expression of L-cell markers. The arrows (A) indicate L-cell marker cytoplasmic staining in normal endocrine cells of associated benign rectal mucosa that serves as a positive control. L-Cell immunohistochemical staining of rectal neuroendocrine neoplasms (NENs) was either focal/localized (B, C) or diffuse (D). L-Cell NEN type is defined by one or more positive markers (immunoscore > 10) (A-D, ×400).
Survival analysis according to L-cell marker expression. Five- and 10-year overall survival rates (OS) in patients with immunoscore group 0 was significantly shorter than that of others (A). The survival curve according to the immunoscore groups strongly supports the reliability of using a cutoff score group of more than 0 for defining the L-cell type (B). (immunoscore group 1: p=0.0061; hazard ratio [HR], 0.226; 95% confidence interval [CI], 0.078 to 0.654; immunoscore group 2: p=0.0059; HR, 0.120; 95% CI, 0.026 to 0.544; immunoscore group 3: p ≤ 0.0001; HR, 0.051; 95% CI, 0.016 to 0.205).
Distribution of L-cell immunoscore groups by tumor grade (A), size (B), extent (C), and lymph node (LN) metastasis (D). Most neuroendocrine tumor (NET) G1 were L-cell type but seven out of 21 NET G2 (33.33%) and three out of 29 neuroendocrine carcinomas (NECs) (10.34%) were in the highest score group 3 for L-cell markers. L-Cell type tumors were rarely found in large size, invasive and even node positive tumor.
The number of rectal NENs from available recorded information by the pathological parameter collected from the nationwide multicenter data
Clinicopahtological parameter | No. of cases with available information (%) | Total | |
---|---|---|---|
Histologic classification (2000 WHO) | WDET | 1,620 (93.37) | 1,732 |
WDEC | 57 (3.29) | ||
PDEC | 45 (2.59) | ||
MEEC | 10 (0.58) | ||
Grade (2010 WHO) | NET G1 | 906 (87.28) | 1,038 |
NET G2 | 66 (6.36) | ||
NEC | 66 (6.36) | ||
Extent | Mucosa-submucosa | 1,582 (92.62) | 1,708 |
Proper muscle | 33 (1.93) | ||
Subserosa | 44 (2.58) | ||
Serosa | 12 (0.70) | ||
Adjacent organ invasion | 4 (0.23) | ||
Distant metastasis | 33 (1.93) | ||
Size (mm) | 1-4 | 547 (38.31) | 1,428 |
5-9 | 604 (42.30) | ||
10-14 | 150 (10.50) | ||
15-20 | 52 (3.64) | ||
> 20 | 75 (5.25) |
WHO, World Health Organization; WDET, well-differentiated endocrine tumor; WDEC, well-differentiated endocrine carcinoma; PDEC, poorly-differentiated endocrine carcinoma; MEEC, mixed exo-endocrine carcinoma; NET G1, neuroendocrine tumor grade 1; NET G2, neuroendocrine tumor grade 2; NEC, neuroendocrine carcinoma.
Correlation analysis of immunoexpression between the three L-cell markers
Glucagon-I | Glucagon-II | PYY | |
---|---|---|---|
Glucagon-I | 1.00000 | - | - |
Glucagon-II | 0.77165 |
1.00000 | - |
PYY | 0.42458 |
0.42876 |
1.00000 |
Pearson correlation coefficient.
Clinicopathologic analysis of rectal NENs in relation to L-cell phenotype
Clinicopathologic parameter | No. of cases | Non–L-cell type | L-Cell type | p-value | |
---|---|---|---|---|---|
Grade | NET G1 | 113 (64.42) | 4 (3.54) | 109 (96.46) | < 0.0001 |
NET G2 | 21 (12.88) | 1 (4.76) | 20 (95.24) | ||
NEC | 29 (17.79) | 17 (58.62) | 12 (41.38) | ||
Total | 163 (100) | 22 (13.50) | 141 (86.50) | ||
Extent | Mucosa/submucosa | 90 (63.38) | 3 (3.33) | 87 (96.67) | < 0.0001 |
Proper muscle | 13 (9.15) | 2 (15.39) | 11 (84.61) | ||
Subserosa | 24 (16.90) | 8 (33.33) | 16 (66.67) | ||
Serosa | 6 (4.23) | 3 (50.00) | 3 (50.00) | ||
Adjacent organ | 2 (1.41) | 1 (50.00) | 1 (50.00) | ||
Distant metastasis | 7 (4.93) | 3 (42.86) | 4 (57.14) | ||
Total | 142 (100) | 20 (14.08) | 122 (85.92) | ||
Size | 141 (100) | 33.6905±26.0809 | 12.4417±12.5830 | < 0.0001 | |
LN metastasis | Positive | 49 (76.56) | 16 (32.65) | 33 (67.35) | 0.1453 |
Negative | 15 (23.44) | 2 (13.33) | 13 (86.67) | ||
Total | 64 (100) | 18 (28.13) | 46 (71.87) |
Values are presented as number (%) or mean±standard deviation. n, number of cases with available data; NET G1, neuroendocrine tumor grade 1; NET G2, neuroendocrine tumor grade 2; NEC, neuroendocrine carcinoma (G3); LN, lymph node.