Endocrine tumors of the gastrointestinal tract and pancreas:grading, tumor size and proliferation index do not predict malignant behavior

Context Gastrointestinal and pancreatic (GIP) endocrine tumors (ETs) have been regarded as slow growing neoplasms with distinct morphologic characteristics that behave less aggressively than carcinomas. The malignant potential of these tumors is difficult to predict. Objective To evaluate prognostic parameters, namely tumor size, tumor grade, and Ki-67 index in relationship to metastatic behavior of GIP ETs. Design Biopsies and surgical specimens from 38 patients with GIP ETs were selected. The study group comprised 16 males and 22 females (mean age 62.6 years; range 24–91). Formalin-fixed, paraffin-embedded tissue sections were stained with H&E, synaptophysin, chromogranin A, and Ki-67. Ki-67 index was evaluated using ChromaVision Automated Assisted Image Analysis software. Proliferative index was compared to tumor grade, and the degree of associations between tumor size, tumor grade, Ki-67 index and metastatic behavior of GIP ETs were evaluated. Results Fifteen of the twenty-two (68.18%) surgically staged neoplasms presented with peritoneal dissemination, lymphogeneous, and/or hematogeneous metastases. Nine of the metastatic tumors were G1 (9/13, or 69.23%), 5 were G2 (5/7, or 71.42%), and 1 – G3 (1/2, or 50%). Overall, 10/15 (66.66%) metastatic tumors showed < 2% Ki-67 immunoreactivity. Four ileal ETs had a synchronous malignancy. No significant correlation was found to exist between tumor grade and Ki-67 index as well as between tumor size, tumor grade, Ki-67 index and metastatic behavior. Conclusion The findings suggest that tumor size, tumor grade and Ki-67 index do not accurately predict malignant behavior of GIP ETs.

The objective of our study is to investigate the potential utility of cell proliferation (Ki-67 index) and histopathologic grading in augmenting the histological classification and assessing biologic aggressiveness in biopsies and surgical specimens with gastrointestinal and pancreatic (GIP) endocrine tumors (ETs).

Classification and histopathologic grading
The tumors were classified according to the World Health Organization guidelines [5]. Histopathologic grading was performed according to the previously published criteria [5,7,8]. Briefly, grade 1 (G1) ETs were characterized by a variable structure, either with insular, trabecular, acinar, diffuse or mixed patterns, and by a monomorphic cytology with low atypia and rare if any mitosis (< 2/10 HPFs). Grade 2 (G2) ETs showed focal moderate cytologic atypia with few scattered mitotic figures (2-10/10 HPFs) and spotty necrosis. Grade 3 (G3) ETs demonstrated a solid growth pattern; the tumor cells were small, round, or oatcell-like with marked nuclear pleomorphism, brisk mitotic activity (> 10/10 HPFs) and sizable areas of tumor necrosis. Degrees of freedom: 2 Chi-square = 0.345368916797488 For significance at the .05 level, chi-square should be greater than or equal to 5.99. The distribution is not significant. p is less than or equal to 1.

Immunohistochemical analysis
Immunostaining was performed according to the manufacturer's specifications. Briefly, four micron-thin sections were placed on the VentanaNexES autostainer (Ventana Medical Systems Inc, Tucson, Arizona) where they were treated with protease for 4 minutes and then incubated with prediluted anti-Ki-67 (Ventana, MM1, mouse monoclonal), anti-synaptophysin (CellMarque, rabbit polyclonal), and anti-chromogranin A (Ventana, LKZH10, mouse monoclonal) for 32 minutes. Recommended positive and negative controls were used. Visualization was performed using Ventana enhanced DAB detection kit. Ki-67 (MM1) stained slides were evaluated using ChromaVision Automated Quantitative Image Analysis software (objective, × 40). All tumor cell areas on the slide that stained positively were included as part of the evaluations regardless of the degree of staining. Cases with Ki-67 immunoreactivity of less than 1% were scored "0".

Statistical analysis
The chi-square test was used to test the association between tumor grade and Ki-67 index as well as between tumor grade, tumor size, Ki-67 index and metastatic behavior of GIP ETs.

Discussion
For many years, GIP ETs have been regarded as slow growing neoplasms with distinct morphologic characteristics that behave less aggressively than conventional adenocarcinomas [1]. The malignant potential of endocrine tumors is difficult to predict. In this context, the latest WHO classification provides a useful framework for the evaluation of clinicopathological and functional properties of these neoplasms [6]. However, a disadvantage of the current WHO classification system is that it is not possible to evaluate some of the well known criteria for malignancy, namely the presence of metastases, and deep wall invasion or invasion of nearby tissue in biopsy specimens. Recently, attempts have been made to define histological and immunohistochemical prognostic factors that may aid in predicting the biologic behavior of GIP ETs in the context in which they commonly present to the surgical pathologist. In this context, the study by Hochwald et al [24] affirmed the clinical usefulness of a Metastatic, G1 endocrine tumor involving ileal mucosa and submucosa Lymph node metastasis from G1 endocrine tumor as shown in Fig. 1
Lymph node metastasis from G1 endocrine tumor as shown in Fig. 1
This study investigated the histological grading, Ki-67 index, tumor size, and metastatic behavior in a group of patients with GIP ETs. The goal was to evaluate Ki-67 index using ChromaVision Automated Image Analysis software, and to determine whether histological grade, tumor size, and Ki-67 index had any bearing on metastatic behavior. We observed unexpectedly high aggressiveness (multiple lymphogenous and hematogenous metastases, and peritoneal implants) in small (< 2 cm), low-grade ETs, with low Ki-67 index. These observations are consistent with other reports [16,18,25]. In his excellent study, based on analysis of 1914 reported cases with gastrointestinal endocrine tumors, Soga [25] found a high aggressiveness in metastasis rates in both rectal and gastric small carcinoids exhibiting values significantly higher than Metastatic, G2 endocrine tumor those of small carcinomas. We did not find statistically significant correlation between tumor grade and Ki-67 index, as well as between tumor grade, tumor size, Ki-67 index and metastatic behavior of GIP ETs. These observations are in disagreement with earlier positive findings [8,11,12,15,19]. This disagreement might be explained by methodological differences, or the different antibodies employed. In this context, ChromaVision Automated Ki-67 index analysis provides superior accuracy in comparison to semi quantitative evaluation of Ki-67 positivity.
Most importantly, this study shows the limitations of the current WHO classification in assessment of the metastatic behavior of GIP ETs. Thus, we were able to show that small, low-grade ETs, with low proliferative index, which met the criteria of the WHO classification criteria for benignity, behaved in a highly aggressive fashion. On the other hand large, intermediate-and high-grade, with high proliferative index ETs, which met the WHO classification criteria for malignancy, behaved in a benign fashion, i.e. without metastatic disease. Currently, we cannot explain the highly aggressive behavior of small, low-grade, with low proliferative index ETs. Previous studies suggest that tumors with a short cell cycle may grow rapidly but without necessarily manifesting numerous mitotic figures at any moment [8]. In addition, recent reports indicate that nuclear survivin and valosin-containing protein (p97) are useful prognostic factors in ETs [26,27].
The observed increased risk of synchronous malignancies in GIP ETs is consistent with previous reports [28][29][30]. The results illustrate the need for a thorough search for additional neoplasms in patients with ileal ETs.

Conclusion
In conclusion, the results suggest that tumor grade does not significantly correlate with Ki-67 index. Further, tumor grade, tumor size, and Ki-67 index do not accurately predict malignant behavior of GIP ETs.