Sporadic Gastric Well-Differentiated Neuroendocrine Tumors Have a Higher Ki-67 Proliferative Index

Well-differentiated neuroendocrine tumor (WDNET) of the stomach can arise in three distinct clinical settings: (1) in association with autoimmune atrophic gastritis, (2) in association with multiple neuroendocrine neoplasia type I (MEN I) or Zollinger-Ellison syndrome (ZES), or (3) sporadic. The Ki-67 proliferative index (PI) in gastric WDNETs in these three distinct clinical settings has not been evaluated in detail. Forty-five gastric WNETs underwent polypectomy (n = 4), endoscopic mucosal resection (n = 12), and surgical resection (n = 29) between 1994 and 2015 were included. H&E slides from each case were reviewed, and Ki-67 immunostain was performed on one representative tumor block. Ki-67 PI was determined by quantitative Aperio image analysis software in areas of strongest nuclear labeling (“hot spots”), and correlated with underlying clinical and pathological features. Twenty-one patients were male and 24 female with a median age of 57 years (range, 30–80 years). Tumors were classified as type I (n = 17), type II (n = 6), and type III (n = 22) WDNETs. Types II and III showed more advanced TNM stage compared to type I (p = 0.02, overall). WHO grade based on Ki-67 PI was higher in type III WDNETs [grade 1 (G1), n = 3; grade 2 (G2), n = 15; and grade 3 (G3), n = 4] than in type I WDNETs [G1, n = 5; G2, n = 12] and in type II WDNETs [G1, n = 2; G2, n = 4] (p = 0.050, overall). Ki-67 PI was significantly higher in type III WDNETs (mean ± SD = 13.0 ± 13.3 %) than in non-sporadic (type I and II) WDNETs (mean ± SD = 5.3 ± 3.3 %; p = 0.015). There was no difference in Ki-67 PI between type I WDNETs (mean ± SD = 5.2 ± 3.5 %) and type II WDNETs (mean ± SD = 5.6 ± 3.1%; p = 0.817). Higher Ki-67 PI was associated with higher tumor T stage (p = 0.003) and also tended to be associated with lymph node metastasis (p = 0.071). In the Kaplan-Meier survival analysis, type I was associated with a significantly longer disease-free survival (DFS) time compared to type II (p = 0.018) or III (0.010). Also, the WHO G3 group had a significantly shorter DFS time than the WHO G1 (p = 0.020) or G2 (p = 0.007) group. Gastric WDNET is a heterogeneous disease entity encompassing three clinical subtypes—type I, type II, and type III—having their own distinct clinicopathologic characteristics and prognosis. Our results showed that sporadic (type III) WDNET had a significantly higher Ki-67 PI than non-sporadic cases (type I or II); increased PI was associated with higher tumor stage. We also described four type III cases of morphologically WD gastric NET with WHO grade 3 on the basis of Ki-67 PI.     

Predicting Prognosis in Gastroentero-Pancreatic Neuroendocrine Tumors: An Overview and the Value of Ki-67 Immunostaining

Gastroenteropancreatic neuroendocrine tumors (GEP-NETS) are unusual and rare neoplasms for which prognostic assessment and the diagnosis of malignancy, on the basis of histology alone, represent considerable challenges for the pathologist. To date, many molecular markers have been identified with a view to providing accurate and timely prediction of response to treatment and long-term survival. Proliferation remains a key feature of tumor progression, which has been widely estimated by the immunohistochemical use of the Ki-67 nuclear antigen. Given the continued difficulties inherent in prediction of malignancy in pancreatic neuroendocrine tumors (PETs) in particular, it has become unclear whether Ki-67 is truly a reliable prognostication marker. This review seeks to better establish what the consensus is on the role of the Ki-67 proliferation index as a prognostic indicator of long-term outcome in pancreatic neuroendocrine tumors. We conclude that most studies favor the utility of the Ki-67 proliferation index despite different critical percentages and in concert with other pathological parameters in the routine work-up of PETs.     

Unraveling Tumor Grading and Genomic Landscape in Lung Neuroendocrine Tumors

Currently, grading in lung neuroendocrine tumors (NETs) is inherently defined by the histological classification based on cell features, mitosis count, and necrosis, for which typical carcinoids (TC) are low-grade malignant tumors with long life expectation, atypical carcinoids (AC) intermediate-grade malignant tumors with more aggressive clinical behavior, and large cell NE carcinomas (LCNEC) and small cell lung carcinomas (SCLC) high-grade malignant tumors with dismal prognosis. While Ki-67 antigen labeling index, highlighting the proportion of proliferating tumor cells, has largely been used in digestive NETs for assessing prognosis and assisting therapy decisions, the same marker does not play an established role in the diagnosis, grading, and prognosis of lung NETs. Next generation sequencing techniques (NGS), thanks to their astonishing ability to process in a shorter timeframe up to billions of DNA strands, are radically revolutionizing our approach to diagnosis and therapy of tumors, including lung cancer. When applied to single genes, panels of genes, exome, or the whole genome by using either frozen or paraffin tissues, NGS techniques increase our understanding of cancer, thus realizing the bases of precision medicine. Data are emerging that TC and AC are mainly altered in chromatin remodeling genes, whereas LCNEC and SCLC are also mutated in cell cycle checkpoint and cell differentiation regulators. A common denominator to all lung NETs is a deregulation of cell proliferation, which represents a biological rationale for morphologic (mitoses and necrosis) and molecular (Ki-67 antigen) parameters to successfully serve as predictors of tumor behavior (i.e., identification of pathological entities with clinical correlation). It is envisaged that a novel grading system in lung NETs based on the combined assessment of mitoses, necrosis, and Ki-67 LI may offer a better stratification of prognostic classes, realizing a bridge between molecular alterations, morphological features, and clinical behavior.     

Molecular Genetic Events in Gastrointestinal and Pancreatic Neuroendocrine Tumors

Gastrointestinal and pancreatic neuroendocrine tumors originate from the cells of the diffuse endocrine system. Their molecular genetic mechanism of development and progression is complex and remains largely unknown, and they are different in genetic composition from the gastrointestinal epithelial tumors. The current literature suggests that multiple genes are involved in their tumorigenesis with significant differences for tumors of different embryological derivatives: foregut, midgut and hindgut. The MEN1 gene is involved in initiation of 33% of foregut gastrointestinal neuroendocrine tumors. 18q defects are present almost exclusively in mid/hindgut neuroendocrine tumors. X-chromosome markers are associated with malignant behavior in foregut tumors only. Analysis of poorly differentiated neuroendocrine carcinomas of any site demonstrates high chromosomal instability and frequent p53 alterations similar to other poorly differentiated carcinomas. Several factors played a limiting role in the molecular studies published to date: the tumors are rare and heterogeneous, it is difficult to predict their behavior and prognosis, and several different tumor classifications are used by the investigators in the studies. Future studies need to evaluate molecular genetic composition of large series of gastrointestinal and pancreatic neuroendocrine tumors of each specific tumor type. Understanding of specific genetic alterations characteristic for gastrointestinal and pancreatic neuroendocrine tumors might lead to their improved diagnosis, morphologic and molecular characterization and treatment.     

Prognostic Significance of p27, Ki-67, and Topoisomerase lla Expression in Clinically Nonfunctioning Pancreatic Endocrine Tumors

Nonfunctioning islet cell tumors or pancreatic endocrine tumors are the most common type of malignant islet cell tumor. Although previously detected usually at an advanced stage because of mass effect, the early detection rate of small localized disease has been increasing. To date it has been difficult to predict the clinical behavior in localized regional nonfunctioning tumors. To investigate potential markers predicting malignancy and poor prognosis in nonfunctioning pancreatic endocrine tumors, we analyzed the expression of Ki-67, topoisomerase IIα (Topollα), and p27, as well as a variety of clinicopathologic parameters in 76 cases of nonfunctioning islet cell tumors (23 benign cases and 53 malignant cases). Ki-67, Topollα, and p27 labeling indices were significantly different between benign and malignant tumors. Expression of Ki-67, Topollα, and p27 were associated with survival in patients with a malignant tumor in a univariate setting. However, only p27 and Topollα were jointly associated with survival in multivariate analysis. Immunohistochemical staining for p27, Topollα, and Ki-67 can be helpful in the diagnosis of nonfunctioning pancreatic endocrine tumor. Analysis of p27 and Topollα may also have potential utility as prognostic factors for malignant tumors.     

The Impact of Phosphohistone-H3-Assisted Mitotic Count and Ki67 Score in the Determination of Tumor Grade and Prediction of Distant Metastasis in Well-Differentiated Pancreatic Neuroendocrine Tumors

This study investigated the impact of phosphohistone-H3 (PHH3)-assisted mitotic count by comparing its performance with conventional mitotic count and Ki67 score as well as the status of distant metastasis. A total of 43 surgically resected pancreatic neuroendocrine tumors (panNET) with complete follow-up information has been subjected to a standardized assessment with respect to mitotic count (both conventional and PHH3-assisted) and Ki67 score. Five participants assessed mitotic count and the time spent was recorded in both methods. All tumors were assigned to a G1 category of mitotic rate on conventional mitotic count that failed to identify three tumors with a G2 category of mitotic rate on PHH3. Near-perfect and fair agreements were achieved among observers when using PHH3 and conventional method, respectively. The mean time spent to determine mitotic count on PHH3-stained slides was significantly shorter (p?<?0.001). The performance of PHH3-assisted mitotic grade category was significant as the three cases with a G2 mitotic category were associated with distant metastasis (p?=?0.01). Despite its performance, the PHH3-assisted mitotic count downgraded 17 cases that were classified as G2 based on Ki67 scores in this series. The Ki67 grade category was either the same or higher than the mitotic grade category. Ten patients developed distant metastasis. Eleven tumors exhibited vascular invasion characterized by intravascular tumor cells admixed with thrombus. Our results indicate that PHH3-assisted mitotic count facilitates an accurate mitotic count with a perfect agreement among observers. The small size of this cohort is an important limitation of the current study, a G2 mitotic grade category based on PHH3 immunohistochemistry was one of the correlates of panNETs with distant metastasis. While the prognostic impact of PHH3-assisted mitotic count needs to be clarified in larger cohorts, Ki67 scores designated higher grade category in all cases; thus, it was the best determinant of the tumor grade. More importantly, the presence of vascular invasion along with the Ki67 grade category was found to be independent predictors of distant metastasis.     

Determination of proliferation index in neoplasms using different Ki-67 equivalent antibodies

Paraffin sections from 23 tumours were immunohistochemically stained with the following four Ki-67 equivalent antibodies: monoclonal MIB-1 (DAKO), monoclonal MM1 (Novocastra), polyclonal NCL-Ki-67p (Novocastra), and polyclonal Rah Ki-67 (DAKO). Ki-67 labelling indices were determined by counting in exactly the same area in each case. MIB-1 showed the highest labelling index in 21 of the 23 cases, and the mean MIB-1 index was approximately 30% higher than that of the other antibodies. The differences between MM1, NCL-Ki-67p and Rah Ki-67 were small and non-significant. There was a positive correlation between each of the four antibodies. As these findings may be of importance when the Ki-67 labelling index is used as a criterion for tumour grading or for clinical prognostication, this necessitate identification of the antibody used in every case.     

Neuroendocrine Tumors of the Breast

A small but increasingly recognized and studied subset of breast carcinomas are characterized by neuroendocrine (NE) differentiation. As with nearly all forms of breast neoplasia, NE tumors are characterized by considerable heterogeneity in microscopic appearance and clinical aggressiveness. About half of NE breast carcinomas recapitulate the histological spectrum typical of their counterparts in other organ systems, varying from “carcinoid-like” to small cell carcinoma, with most representing intermediate grade tumors. Despite NE morphology, these tumors have a high frequency of estrogen receptor expression. Clinical outcomes of women with NE breast carcinomas are reliably grade and stage dependent. Tumors associated with “solid papillary” differentiation comprise the remaining cases of NE breast neoplasia. Solid papillary carcinoma is an intrinsically low grade/favorable prognosis class of breast neoplasia that usually presents in post-menopausal age groups. About half of solid papillary carcinoma present as a distinctive pattern of ductal carcinoma in situ that may be difficult to recognize owing to its close resemblance to florid proliferative lesions. Invasive solid papillary carcinomas are characterized by a variety of histological patterns and often show mucinous differentiation. Future studies are necessary to better define the histogenesis, optimal classification, and improved directed therapies for NE breast neoplasia.     

Comparison of Ki-67 equivalent antibodies

AIMS: To compare commercially available Ki-67 equivalent antibodies with regard to qualitative and quantitative immunohistochemical staining characteristics. METHODS: The following antibodies were used: monoclonal MIB-1 (Immunotech), monoclonal MM1 (Novocastra), polyclonal NCL-Ki-67p (Novocastra), and polyclonal Rah Ki-67 (Dako). All immunostainings were evaluated in squamous epithelium from formalin fixed and paraffin wax embedded pharyngeal tonsils. Labelling indices (LIs) were recorded twice to test their reproducibility. RESULTS: By application of all four antibodies the nuclear staining could be either diffuse, granular, or a combination of both (classified as granular in this study). The diffuse pattern generally showed a strong or moderate staining intensity, whereas the granular pattern displayed a continuum from strong to very weak, making it difficult to discriminate between positive and negative nuclei. The diffuse staining pattern was seen in approximately 59% of the nuclei with the MIB-1 antibody and in 35-45% when the other antibodies were used. The following mean LIs were recorded: MIB-1, 31%; NCL-Ki-67p, 21%; Rah Ki-67, 17%; and MM1, 14%. The reproducibility was excellent for all four antibodies, with the mean of differences between the two runs of counts ranging from 1.1% to 1.5%. CONCLUSIONS: The four tested Ki-67 equivalent antibodies revealed differences in qualitative and quantitative staining characteristics, which resulted in considerable variations in registered LIs. The MIB-1 antibody appears to have a higher sensitivity for detecting the Ki-67 antigen than the other three tested antibodies. These differences are important to consider when proliferative activity is determined by the Ki-67 LI.     

Overview of Neuroendocrine Cells and Tumors

The diffuse neuroendocrine system (DNES) is composed of cells and tumors with secretory granules ranging from 50–400 nm in diameter. Members of the DNES commonly stain for chromogranin and synaptophysin by immunohistochemistry and may express a variety of peptide hormones. Recent studies have shown that the proprotein convertases (proconvertases or PCs) are good broad-spectrum markers for members of the DNES. Gene expression can be readily detecting in neuroendocrine cells and tumors byin situ hybridization (ISH). Newer techniques such asin situ polymerase chain reaction (PCR) can be used to detect gene products that are expressed in low copy numbers in neuroendocrine cells. The concept of multidirectional differentiation is an important notion that helps to explain multiple patterns of phenotypic expression observed in some neuroendocrine tumors. Presented at the Endocrine Pathology Society—USCAP Meeting, Washington, DC, March 23, 1996.     

New Genetics and Genomic Data on Pancreatic Neuroendocrine Tumors: Implications for Diagnosis,Treatment, and Targeted Therapies

The recent findings on the roles of death-associated protein 6/α-thalassemia/mental retardation X-linked (DAXX/ATRX) in the development of pancreatic neuroendocrine tumors (PanNETs) have led to major advances in the molecular understanding of these rare tumors and open up completely new therapeutic windows. This overview aims at giving a simplified view on these findings and their possible therapeutic implications. The importance of epigenetic changes in PanNET is also underlined by recent findings of a cross-species study on microRNA (miRNA) and messenger RNA (mRNA) profiles in PanNETs.     

Nuclear Accumulation of B-Catenin in Human Endocrine Tumors: Association with Ki-67 (MIB-1) Proliferative Activity

β-Catenin is closely associated with carcinoma invasion/metastasis and poor survival. Recent studies have demonstrated that abnormal expression of β-catenin, especially its nuclear accumulation, also plays an important role in wingless/Wnt signaling pathway. In this study, we evaluated immunohistochemically the nuclear localization of β-catenin in a total of 93 human-endocrine-related tumors including 1 medullary carcinoma (thyroid gland), 12 parathyroid tumors, 22 carcinoid tumors (digestive tract and liver), 7 islet cell tumors, 26 adrenocortical tumors, 13 neuroblastoma (adrenal gland), and 12 pheochromocytoma (adrenal gland), and also studied genetic alterations of the β-catenin gene. Nuclear accumulation of β-catenin was frequently detected in 8 of 22 (36%) carcinoid tumors and 2 of 7 (29%) islet cell tumors. No genetic alteration in exon 3 of the β-catenin gene encoding serine/threonine rich domain, which was phosphorylated by GSK-3β, was detected in any groups of the endocrine tumors. However, nuclear accumulation of β-catenin in carcinoid tumors was significantly correlated with the proliferative marker Ki-67 (MIB-1) labeling index (p<0.001). Our findings suggest that nuclear transfer and accumulation of the β-catenin may contribute in the tumorigenesis of carcinoid tumor as an oncoprotein.     

Ki-67 reactivity in primary fallopian tube cancers

Forty-four cases of primary cancer of the fallopian tube (PFTC) were analyzed as to Ki-67 expression, grade, stage and the cancer histological type. Among patients with an average age of 57.5 years (range 38-70 years), 27 patients were FIGO I, 7 were FIGO II and 10 were FIGO III. Histological classification of PFTC revealed 18 cases of endometrioid type, 9 serous, 7 undifferentiated, 6 urothelial, 2 clear-cell and 2 of other type. Histological grading revealed 11 cases of G1, 16 of G2 and 17 of G3 tumors. The quantity of Ki-67 positive cells was counted on 300 cancer cells in random high-power fields (10 x 40) and recorded as the labeling index (LI, %). Positive staining for Ki-67 was shown in the nuclei in all cases. Ki-67 LI values ranged from 14.2 to 97.2% (median 36.1). Ki-67 LI values were graded as > or = 36.1% as high and <36.1% as low. We did not find any significant differences in Ki-67 LI values among tumors of various clinical stages, histological grades and histological types. The p value was statistically significant only for stage as a prognostic factor.     

胃肠道间质瘤中p53、Ki-67、Bcl-2、CyclinD_1的表达及其与预后的关系

目的:分析32例胃肠道间质瘤(GISTs)病人组织切片中p53、Ki-67、Bcl-2和CyclinD1表达及其与预后的相关性。方法:采用免疫组化SABC法检测32例GISTs患者p53、Ki-67、Bcl-2和CyclinD1的表达。结果:p53在良性组均呈阴性,在交界组与恶性组呈阳性者为3例和15例;Ki-67指数在三组中表达分别为(8.97±4.12)%、(16.21±7.44)%和(22.33±9.14)%(P<0.01);Bcl-2在三组呈阳性者分别为2例、3例和12例;CyclinD1指数在三组中表达分别为16.95%、51.22%和69.38%。Ki-67表达大于20%有12例,存活时间平均10.5月,Ki-67表达小于20%的存活时间平均22.4月。结论:Ki-67增值指数不仅可用作评价GISTs恶性潜能的指标,而且Ki-67指数大于20%具有独立的预后意义。     

Endocrine Precursor Lesions of Gastroenteropancreatic Neuroendocrine Tumors

This review focuses on precursor lesions of gastrointestinal and pancreatic neuroendocrine tumors (GEP-NETs). There are three conditions that are associated with hyperplastic changes in endocrine cells preceding GEP-NETs: autoimmune chronic atrophic gastritis or multiple endocrine neoplasia type 1 (MEN1) with gastric enterochromaffin-like (ECL) cell hyperplasia; MEN1 with gastrin and somatostatin cell hyperplasia in the duodenum and glucagon cell hyperplasia in the islets of the pancreas; and inflammatory bowel disease with endocrine cell hyperplasia in the colon. In gastric ECL cell hyperplasia, it is assumed that hypergastrinemia promotes the growth of the ECL cells of the corpus mucosa and leads to hyperplasia and neoplasia. In the duodenum and the pancreas, the MEN1-associated germline mutation of the menin gene obviously causes hyperplasia of the gastrin and somatostatin cells (duodenum) and the glucagon cells (pancreas), resulting in multifocal development of tumors. These tumors show allelic deletion of the MEN1 gene, whereas the precursor lesions retain their heterozygosity. The endocrine cell hyperplasia in the colon described in inflammatory bowel disease has neither a genetic nor a definite hormonal background.     

Ki-67 and biological behaviour in meningeal haemangiopericytomas

The biological behaviour of meningeal haemangiopericytomas was retrospectively studied using immunohistochemical staining with MIB1, a monoclonal antibody against the Ki-67 antigen, a nuclear protein related to cell proliferation. Paraffin-embedded material from 62 tumours from 40 patients were investigated. The proliferating compartment of the tumours was estimated by evaluating the MIB1 staining index, i.e. the percentage of MIB1 positive nuclei in at least 1000 counted tumour cells in representative areas. The staining index ranged from 1.24% to 39.01%. Statistical analysis revealed no significant correlation between the staining index and recurrence-free survival (χ² = 0.3922, P  = 0.5311). Long-term observation (>100 months), however, revealed a tendency to longer survival in the group with a staining index less than 5%. According to our results, the MIB1 staining index does not contribute to the accuracy of predicting the clinical outcome of meningeal haemangiopericytomas.     

Cathepsin D Inhibits Angiogenesis in Pituitary Neuroendocrine Tumors

Prolactin and growth hormone can acquire anti-angiogenic properties after undergoing proteolytic cleavage by Cathepsin D and bone morphogenetic protein 1 (BMP-1) into fragments known as vasoinhibins. Little is known about the effect of vasoinhibins on angiogenesis through the involvement of key cleavage enzymes Cathepsin D and BMP-1 in pituitary neuroendocrine tumors (PitNETs, formerly pituitary adenomas). The purpose of this study was to investigate the mechanism of action of Cathepsin D and BMP-1 on angiogenesis in PitNETs compared with that of pro-angiogenic factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-2 (FGF2). A total of 43 patients were enrolled in a retrospective analysis and 22 samples were suitable for RNA extraction, including 16 nonfunctional PitNETs and six somatotroph tumors. The mRNA and protein levels of Cathepsin D, BMP-1, VEGF, and FGF2 were compared with those of von Willebrand factor, which was assessed to determine the vascularization of PitNETs. Cathepsin D and FGF2 were significantly correlated with vascularization in PitNETs. Both Cathepsin D and FGF2 are highly involved in angiogenesis in PitNETs, although the effect of Cathepsin D as an anti-angiogenic factor is dominant over that of FGF2 as a pro-angiogenic factor.     

Reliability of Criteria for Ultrastructural Identification of Neuroendocrine Granules

For full diagnostic use to be made of the neurosecretory granule, the range of sizes, forms, and staining qualities for this cytoplasmic organelle, along with the extent of its expression in various neoplasms, must be established. Neurosecretory type granules occasionally occur in nonneuroendo-crine tumors. A series of carcinoids of the lung provides a model for assessing the morphologic types of cytoplasmic granules identified by antibodies to chromogranin A and immunogold labeling. The results show that granule structure in tumors is pleomorphic. Despite having sizes within the expected range, many labeled and, indeed, unlabeled secretory granules are atypical, particularly in structural form. Cell-to-cell variation in the proportion of even typical neurosecreotry granules labeling for chromogranin A is the rule. Studies correlating biochemical, immunohistochemical, electron microscopic, and perhaps in situ hybridization characteristics are required to define better the criteria for unequivocal identification of neurosecretory granules in tumors.