Prognostic and biological significance of cytokeratin 19 in pancreatic endocrine tumours

AIM: The prognosis of well-differentiated pancreatic endocrine tumours (PETs) is difficult to establish on a histological basis. The expression of cytokeratin (CK19) has recently been proposed as an indicator of unfavourable outcome. However, this finding still needs to be verified and to be compared with more frequently used prognosticators such as proliferative indices, vascular and/or perineural invasion. The aim of this study was to evaluate the prognostic value of CK19 expression in PETs. METHODS AND RESULTS: One hundred and forty-five PETs were studied using two different anti-CK19 monoclonal antibodies (BA17 and RCK108). The results were statistically compared with proliferation markers, vascular and perineural invasion and the presence of metastases. On univariate analysis, CK19 immunoreactivity correlated with prognosis only when it was detected with the RCK108 antibody and only in the whole group of PETs and in insulinomas. Conversely, it did not predict survival in non-functioning neoplasms. Ki67 index, mitotic count, vascular and perineural invasion were all statistically correlated with prognosis. On multivariate analysis, only the Ki67 index and metastases were independent prognosticators. CONCLUSIONS: CK19 expression correlates with patient survival only when detected with the RCK108 antibody and mainly in insulinomas. Ki67 index and metastases represent the only two independent predictors of survival.     

Prognostic criteria in nonfunctioning pancreatic endocrine tumours

To identify prognostic subgroups among nonfunctioning (nonsyndromic) pancreatic endocrine tumours, a series of 61 tumours were analysed systematically for macroscopic, histopathological and immunohistochemical variables potentially predictive of malignancy. High-grade nuclear atypia, elevated mitotic rate and multifocal necrosis allowed us to separate 5 poorly differentiated carcinomas from 56 well differentiated tumours. Among the latter, 29 well-differentiated carcinomas showing gross local invasion or metastases were identified. Vascular or perineural microinvasion, Ki67 proliferative index >2%, mitotic rate 2, size 4 cm, capsular penetration, nuclear atypia, lack of progesterone receptors and presence of calcitonin were among the variables correlated with malignancy. The first two were the most sensitive and specific. Their presence or absence was used in the 27 tumours lacking evidence of malignancy at the time of surgery to separate 11 cases with increased risk of malignancy (in 2 of which metastases developed during follow-up) from 16 cases with limited risk. The resulting four prognostic groups of nonfunctioning pancreatic endocrine tumours (limited- and increased-risk tumours, well-differentiated carcinomas and poorly differentiated carcinomas) showed distinct survival curves, which were significantly affected by vascular microinvasion, Ki67 proliferative index and metastases.     

Malignant potential of aneuploid pancreatic endocrine tumours

Evaluation of the malignant potential of pancreatic endocrine tumours is difficult by histological criteria alone. Nuclear deoxyribonucleic acid (DNA) cytometric analysis can provide significant prognostic and biological information in a number of solid human tumours. Thus, the DNA profiles and nuclear parameters of 39 patients with pancreatic endocrine tumours diagnosed from 1969 to 1989 were studied, using computerized video image analysis. Of the 39 patients with tumours, 27 cases did not have evidence of metastatic disease at the time of investigation. The majority of these (N=21) showed a diploid profile. Five were aneuploid and one was tetraploid. In the 12 tumours that proved malignant, three had tissue of both the primary tumour and the metastases analysed; six had material only from the primary tumour, although metastatic disease was established clinically; and another three only had tissue from the secondary tumour studied. The majority of the malignant cases were aneupioid (N =7); three were dipioid; one showed a diploid primary but an aneuploid secondary; and one showed a hyperdiploid pattern. The percentage of nuclei with a DNA content greater than 5N (5CER) did not seem to contribute further to the assessment nor did the means of the nuclear areas and shape factors. It appears that ploidy studies may be useful in predicting malignant potential of pancreatic endocrine tumours. There is a high rate of immunostaining for insulin by benign pancreatic endocrine tumours and this may be a useful adjunct to distinguish them from their malignant counterparts. However, there is no statistically significant correlation between the expression of various hormones and the DNA ploidy of tumour cells.     

Prevalence of CD99 protein expression in pancreatic endocrine tumours (PETs)

AIMS: To determine the prevalence of CD99 expression in pancreatic endocrine tumours (PETs). We evaluated CD99 expression and analysed Ki67 labelling by immunohistochemistry in PETs. METHODS AND RESULTS: Thirty-eight PETs from 33 patients were analysed. CD99 immunoreactivity was consistently observed in normal islets of the pancreas, regardless of the cell type. Tumours comprising more than 30% CD99+ cells were defined as positively immunoreactive for CD99. CD99 expression was observed in 20 of the 38 PETs examined, but not in any of the pancreatic tumours of other histological subtypes (10 ductal adenocarcinomas, five intraductal papillary-mucinous tumours, and two acinar cell tumours). Loss of CD99 expression was related to markers of worse prognosis for PET, including gross local invasion, metastasis to the lymph nodes or other organs, lymphatic or blood vessel invasion, and neuroendocrine carcinoma (NEC). Thus, CD99 expression may have an efficiency comparable to that of high Ki67 labelling index (5% or more) for prognostication. CONCLUSIONS: CD99 expression was observed frequently and exclusively in PETs, and loss of CD99 expression in PETs was found to be associated with ominous prognostic indicators.     

OVEREXPRESSION OF THE 67-kD LAMININ RECEPTOR CORRELATES WITH TUMOUR PROGRESSION IN HUMAN COLORECTAL CARCINOMA

The high affinity 67-kD laminin receptor (67LR) is a cell surface protein whose expression is increased in a number of human carcinoma models. To date, 67LR expression in colorectal carcinomas has been examined in a small number of cases. 67LR expression has been immunohistochemically analysed in a large series of human colorectal neoplasms, using the MLuC5 monoclonal antibody. The study included 59 samples of non-neoplastic mucosa, 45 polyps (11 hyperplastic, 34 adenomas), 196 carcinomas, and lymph node metastases of 87 carcinomas. Epithelial cells of normal mucosa and hyperplastic polyps were negative or showed weak positivity in the paranuclear and apical areas of the cytoplasm. In adenomas and carcinomas, the staining was stronger, with a membranous or cytoplasmic pattern. The expression of 67LR correlated significantly with the progression from normal mucosa (22 per cent) to adenoma (44 per cent), carcinoma (61 per cent), and lymph node metastasis (75 per cent) ( P <0·0001). Expression of the laminin receptor showed a tendency to be more frequently positive in advanced stage (III+IV; 67 per cent) when compared with early stage (I+II) carcinomas (54 per cent). The difference, however, was not statistically significant ( P =0·058). In addition, 14 out of 28 (50 per cent) primary carcinomas without 67LR expression became positive in lymph node metastases, while most (86 per cent) of the MLuC5-positive primary carcinomas were also immunoreactive in metastases. In conclusion, these results indicate that 67LR is up-regulated in the progression of human colorectal carcinomas and may play a role in the local and metastatic progression of these tumours.     

Neuropilin-2 is a novel marker expressed in pancreatic islet cells and endocrine pancreatic tumours

Neuropilin-2 (NP-2) is a cell surface transmembrane protein originally characterized as a receptor for the type 3 semaphorins, and more recently for a number of vascular endothelial growth factor (VEGF) isoforms. NP-2 expression has been recently localized to a subset of neuroendocrine cells in the gastrointestinal tract. The aim of this study was to define the expression pattern of NP-2 in normal pancreatic islets and to determine the utility of NP-2 expression as a diagnostic marker of pancreatic endocrine tumours. Paraffin-embedded tissue sections from 30 endocrine pancreatic tumours (EPTs) and from normal pancreas were immunostained with a rabbit polyclonal antibody generated towards NP-2. Nineteen of the tumours were hormonally functional (nine insulinomas, nine gastrinomas, and one glucagonoma). The NP-2 staining pattern was correlated with islet cell hormone expression. In addition, NP-2 expression was evaluated in other normal neuroendocrine tissues and neuroendocrine neoplasms. In normal pancreas, NP-2 stained a distinct subset of islet cells situated primarily at the islet periphery. Double immunohistochemical staining revealed co-localization with glucagon-expressing cells. Moderate to strong NP-2 staining was present in 27 of 30 EPTs. Serial staining of the pancreatic tumours with insulin, gastrin, glucagon, pancreatic polypeptide (PP) or somatostatin did not reveal a distinct pattern of co-localization. NP-2 expression was not detected in neuroendocrine cells outside the gastroenteropancreatic system, or in their corresponding neoplasms, except for focal staining in one bronchial carcinoid tumour. In conclusion, the vast majority of EPTs examined expressed NP-2, suggesting its utility as a diagnostic marker for these tumours. The function of NP-2 in islet cell biology or tumourigenesis remains to be elucidated.     

Morphology and immunohistochemically-defined endocrine function of pancreatic islet cell tumours

Thirty pancreatic islet cell tumours were histologically classified and analysed for their possible peptide hormone content using the immunohistoperoxidase method. Seven tumours contained insulin, six tumours contained gastrin and eight tumours contained glucagon. One tumour contained all three hormones. In the insulin and gastrin-containing tumours, the cells were usually arranged in solid nests of cells, with tubular and acinar formations in about half the cases. In the glucagon-containing tumours the cells were mainly arranged in anastomosing ribbons consisting of one of two layers of small cells. Most of the hormone-containing tumours were argyrophilic using Grimelius' silver reaction. All but one of the glucagon-containing tumours were incidental findings at autopsy. About half of the other tumours had metastasized. It is concluded that a relation exists between the histological pattern of growth and immunohistochemically defined endocrine function of pancreatic islet cell tumours.     

THE SPECTRUM OF 67-kD LAMININ RECEPTOR EXPRESSION IN BREAST CARCINOMA PROGRESSION

Laminin is a glycoprotein of the basement membrane (BM), involved in a variety of normal and pathological cellular events including tumour invasion and metastasis. Cells bind laminin through different types of receptor. The 67-kD laminin receptor (67LR) is a cell-surface protein which binds laminin with high affinity. 67LR expression has been shown to increase in neoplastic cells, compared with normal tissues, and 67LR seems to play an important role during the first steps of neoplastic progression. In this study, 67LR expression was analysed during the morphological phases of breast cancer progression from normal tissue to invasive carcinoma. A total of 506 formalin-fixed, paraffin-embedded normal breast structures and lesions were stained by immunohistochemistry using the MLuC5 monoclonal antibody, which is specific for 67LR. The results show that in normal breast and in any kind of breast lesion, myoepithelial and endothelial cells express 67LR. While 67LR is not seen in the epithelium of normal breast, cysts, adenosis, and benign tumours, it is expressed in the epithelial cells of several hyperplasias and carcinomas in situ, both ductal and lobular, as well as in all invasive carcinomas. The 67LR-positive cell subpopulation expands from hyperplastic lesions to invasive carcinoma, suggesting that 67LR could be related to the induction and progression of breast cancer. © 1997 John Wiley & Sons, Ltd.     

Prognostic and predictive factors in endocrine tumours

This review encompasses the diagnostic features of malignancy, the routinely observable prognostic features and the prognostic and predictive features emerging from research techniques in the principal endocrine neoplasms: pancreatic and extrapancreatic endocrine cell tumours, thyroid and parathyroid neoplasia, adrenal cortical neoplasms and adrenal and extra-adrenal paragangliomas. While each endocrine tissue has its own set of diagnostic features for malignancy, and prognostic features once a diagnosis of malignancy has been established, there are a few common themes. For several endocrine neoplasms, definite recognition of malignancy can be difficult and may depend upon frank invasive or metastatic growth at presentation. Endocrine tissues are dynamic, with hyperplastic and regressive phenomena, some of which may mimic malignancy. Even when unequivocal features of malignancy are available for observation, their distribution in tissue may be very focal, necessitating thorough sampling. The accurate documentation of routinely observable histological features interpreted in the light of current literature has not been superseded by special techniques in the statement of diagnosis or prognosis in the vast majority of endocrine neoplasms.     

The endocrine cells of the pancreas and related tumours

Summary Up to seven endocrine cell types have been identified ultrastructurally in the pancreas, including glucagon A cells, insulin B cells, somatostatin D cells, pancreatic peptide F cells and 5-hydroxytryptamine EC cells. In addition, D₁ cells, which have been proposed as the cell type producing VIP and possible P cells of unknown function are seen. Various patterns of endocrine cell differentiation have been found in 20 endocrine pancreatic tumours. Well and poorly differentiated B cells have been identified in 6 insulinomas, diagnostic G cells in 3 out of 7 gastrinomas, D₁ and/or F cells in 7 diarrheogenic tumours. Moreover, cells apparently unrelated to the prevalent clinical syndrome have been noted in 8 of the 20 tumours. Granular non diagnostic cells (poorly diagnostic gastrin cells? D₁ cells?) were particularly frequent in gastrinomas; agranular or poorly granular cells, either of active or stem cell type, were present in nearly all tumours, particularly in diarrheogenic tumours, gastrinomas and malignant insulinomas. A cytological classification of pancreatic endocrine tumours is proposed.     

Pathological assessment of pancreatic endocrine tumors for metastatic potential and clinical prognosis

The prognostic significance of several pathological factors (tumor size, mitotic index, Ki-67 labeling index, and vascular invasion) and expression of exocrine markers (CA19-9, CEA, AFP, and trypsin) in pancreatic endocrine tumors was studied. A total of 20 specimens of metastasizing (n=10) and non-metastasizing (n=10) tumors were subjected to histological and immunohistochemical examination. The metastasizing tumors showed significantly larger size, higher Ki-67 labeling index, increased number of mitotic cells, and more frequent vascular invasion in comparison with the non-metastasizing tumors. It was difficult to determine the effect of individual factors on clinical outcome because of slow disease progression in almost all cases. Numerous mitotic cells and widespread necrosis, however, were thought to indicate a poor prognosis, and tumors with these characteristics were regarded as high-grade malignant endocrine carcinomas. In one case, one-third of the tumor tissue comprised trypsin-positive cells, the outcome was comparatively poor, and the behavior of the tumor resembled that of mixed acinar-endocrine carcinoma. A simple multifactorial approach may be effective for the identification of tumors at increased risk of metastasis, but it remains difficult to determine clinical prognosis. It is essential to at least distinguish high-grade endocrine carcinomas from the more common endocrine tumors.     

Oncoprotein immunoreactivity in human endocrine tumours.

Abnormally expressed oncogenes are implicated in neoplastic transformation. We have investigated a series of endocrine tumours using immunocytochemistry as a first screening tool to detect oncogene expression. Paraffin sections of 44 pulmonary small cell carcinomas, 15 pulmonary atypical carcinoids, 12 bronchial carcinoids, 28 medullary thyroid carcinomas, 27 phaeochromocytomas, and 17 insulinomas were immunostained with antibodies to c-erbB-2, c-myc, L-myc, and N-myc. Diffuse immunoreactivity was detectable for c-erbB-2 in 8 out of 44 (18 per cent) pulmonary small cell carcinomas, 3 out of 15 (20 per cent) pulmonary atypical carcinoids, and 6 out of 27 (22 per cent) phaeochromocytomas; for c-myc in 18 out of 44 (41 per cent) pulmonary small cell carcinomas and 5 out of 15 (33 per cent) pulmonary atypical carcinoids; for N-myc in 6 out of 28 (21 per cent) medullary thyroid carcinomas; and for L-myc in 4 out of 27 (15 per cent) phaeochromocytomas. There was considerable intratumoral and intertumoral heterogeneity and, in each tumour group, no relationship was found between tumour pattern, mitotic index, and oncoprotein immunoreactivity. These results suggest that oncogene products are present in a proportion of endocrine tumours, and that specific oncoproteins seem to be related to tumour type but not to other histopathological findings. Thus, oncoprotein detection may be a useful tool for identifying subsets of endocrine tumours that are not otherwise recognizable morphologically.     

Characterization of two monoclonal antibodies directed against the 67 kDa high affinity laminin receptor and application for the study of breast carcinoma progression

Two monoclonal antibodies (mAbs), designated MLuC5 and MLuC6, were produced against a human small cell lung carcinoma cell line. They were found to exhibit a superimposable reactivity on different cell lines and on platelets. Moreover, they both immunoprecipitated a 67 kDa molecule from the membrane of the reference target cells. Immunodepletion and cross-inhibition tests indicated that the two mAbs recognize two epitopes closely localized on the same molecule. The MLuC5 mAb was further characterized for its reactivity on platelets. Immunoprecipitation and ELISA assays demonstrate that this mAb recognizes the 67 kDa high afinity laminin receptor. MLuC5 reactivity was evaluated by immunohistochemistry on a variety of normal and tumor tissues, in particular breast specimens including normal epithelium, dysplastic lesions, in situ carcinomas, invasive primary carcinomas and distant metastases. The laminin receptor was found to be strongly expressed in 50% of the infiltrating carcinomas, whereas in situ carcinomas and benign lesions, as well as the normal mammary epithelium, were only weakly and focally positive. In metastatic lesions MLuC5 reactivity was only found in 11% of the samples tested, independently of the site of origin of the lesion.     

Nucleolar organizer regions and glycoprotein-hormone α-chain reaction as markers of malignancy in endocrine tumours of the pancreas

The value of silver staining of nucleolar organizer regions (AgNORs) and human chorionic gonadotropin α-chain reaction (HCG-α) as markers of malignancy was investigated in 60 primary pancreatic endocrine tumours, 37 of which had metastasized at the time of surgery, and in one of which metastases developed 4 years after surgery. Assessment of AgNORs by digital image analysis revealed few but large AgNORs (mean number 2.5 ± 1.1; mean area ± in the 22 benign tumours and many but small AgNORs (mean number 5.1 ± 1.9, P <0.05; mean ± 9 μm². P <0.01) in the malignant tumours. Quantification of the number of AgNORs per tumour cell AgNOR distribution score) showed that 96% (26/27) of tumours exhibiting at least 5% of cells with more six AgNORs per nucleolus showed metastases either at the time of diagnosis or up to 4 years after surgery. HCG-α immunoreactive cells were present in 25/38 (66%) malignant tumours and in 4/22 (18%) benign tumours. Combined evaluation of AgNOR distribution and HCG-α scores showed a high positive predictive value of 96% in cases with a raised AgNOR distribution score irrespective of the HCG-α status. A good negative predictive value (81%) was, however, only obtained if both parameters, AgNOR distribution and HCG-α scores, were negative. Thus, investigation of AgNORs HCG-α is helpful in predicting malignancy in a high percentage of pancreatic endocrine tumours.     

Expression of the 67 kD laminin receptor in human cervical preneoplastic and neoplastic squamous epithelial lesions: an immunohistochemical study

Interactions of cancer cells with laminin play a critical role during the progression of solid malignant tumours. Increased expression of the 67 kD laminin receptor (67LR), one of the several laminin binding proteins, is associated with the invasive and metastatic capacity of various types of cancer, including breast, colon, ovary, lung, and endometrial carcinoma. In this study, 67LR expression was analysed in a series of cervical biopsy specimens including 16 normal cervical tissues, 36 low-grade squamous intraepithelial lesions (SILs), 24 high-grade SILs, and 11 invasive carcinomas. Detection of the 67LR was performed using immunoperoxidase staining and the monoclonal antibody MLuC5 which specifically recognizes the 67LR. Immunostaining of the 67LR was correlated with human papillomavirus (HPV) type detected by in situ hybridization and with proliferative activity of the lesion determined by immunohistochemistry with the MIB-1 monoclonal antibody, specific for the Ki67 antigen. Increased expression of the 67LR was correlated with the histological severity of the lesions, with the strongest immunoreactivity being found in invasive carcinomas. Significant differences in 67LR expression were found between normal cervical epithelium and high-grade SILs (P<0·05, non-parametric Mann-Whitney test) or invasive carcinomas (P<0·001), as well as between low- or high-grade SILs and invasive carcinoma (P<0·01 and P<0·05, respectively). Ki67 antigen expression also increased with the severity of the lesions. There was a positive correlation for each type of lesion between expression of the 67LR and of the Ki67 antigen. No specific relationship was found between 67LR or Ki67 antigen immunostaining and HPV type detected in SILs, segregated into low-grade and high-grade lesions. These data add weight to the evidence that increased expression of the 67LR is a consistent, but not sufficient feature of the invasive and metastatic phenotype and suggest that high expression of the 67LR might be associated with both more proliferative and more aggressive cervical (pre)neoplastic lesions. © 1997 John Wiley & Sons, Ltd.     

Control pathways of the 67 kDa laminin binding protein: surface expression and activity of a new ligand binding domain

A number of papers have been published on the clinical correlation of the expression of the 67 kDa laminin binding protein (LBP) with the metastatic potential of solid tumors. Both mRNA and protein expression levels have been reported, but both the relationship between them and the molecular nature of the 67 kDa surface product remain unclear. We have utilized a homotypic overexpression system to investigate the cell surface presentation of the 67 kDa LBP and the contribution of this protein to the invasive phenotype of cultured cell lines. We report here that the cellular mRNA levels do not directly reflect the levels of the 67 kDa LBP observed on the cell surface in this overexpression system. Methotrexate amplification of transfected plasmids expressing the 67 kDa LBP leads to an initial elevation of both the LBP mRNA and surface protein levels. This is accompanied by an altered, more flattened, cell morphology. Later, apparent adaptation of the cells to methotrexate is accompanied by a down-regulation of the surface expression of the protein. mRNA levels, however, remain elevated. A nine amino acid sequence, CDPGYIGSR (peptide 11), within the chain of laminin 1 has been identified as a probable binding domain for the 67 kDa LBP. Previous studies have identified a region of the 67 kDa LBP which may be involved in laminin interaction, although not necessarily via the peptide 11 domain. We have identified a second site within the amino acid coding sequence of the 67 kDa LBP which also shows biological activity both in vitro and in vivo. A peptide with this sequence, LBP residues 205-229, binds laminin-1 in a peptide 11 inhibitable manner. The receptor-derived peptide modulates invasion of basement membrane matrix in vitro and inhibits experimental lung colony formation when injected along with B16BL6 mouse melanoma cells. However, pretreatment of the melanoma cells with the peptide enhances lung colony formation. Thus, the interaction of the 67 kDa LBP with basement membrane matrix appears to involve a complex series of events including multiple adhesive sites and tight regulation of cell surface expression.     

Criteria for malignancy in gastrointestinal endocrine tumors

In contrast with the large amount of data generated from endocrine tumors of the pancreas, sparse and mostly unconfirmed data are available on the criteria for the assessment of malignancy risk and patient outcome in endocrine tumors of the gastrointestinal tract. In these conditions the 2000 WHO classification with its standardized scheme of pathologic report constitutes a framework facilitating the assessment of tumor malignancy and has been regarded as useful for clinical purposes, providing the basis for proper management of the patients and for the design of treatment protocols. The classification is based on a combination of pathological and clinical features with parameters specific for each organ in which the endocrine tumors originate. Three main categories, one further subdivided into two subgroups, are considered: (1) well-differentiated endocrine tumors, further subdivided into tumors with benign and with uncertain behavior; (2) well-differentiated endocrine carcinomas, low grade; and (3) poorly differentiated endocrine carcinomas, high grade. In this review the differential tumor characteristics between the different categories are summarized. Moreover, the relevance of additional features with respect to tumor prognostication, chiefly the Ki-67 proliferation index and malignancy-associated genetic changes, is discussed with emphasis on the discrepancies emerging between tumors of foregut and of midgut origin.     

胃肠道类癌中Ki-67、PCNA、Laminin的表达及意义

目的 研究胃肠道类癌Ki-67、PCNA、laminin(LN)的表达与侵袭性和增殖性的关系。方法 应用免疫组织化学S-P法检测36例胃肠道类癌组织中3种指标的表达。结果 36例胃肠道类癌组织中Ki～67、PCNA、LN阳性表达率分别为50．o％、55．5％、55．5％；随肿瘤分化程度降低、浸润深度加深、淋巴结的转移，各指标阳性表达率逐渐升高。结论 Ki-67、PC-NA可作为评估胃肠道类癌预后的指标。Ki-67比PCNA在评价胃肠道类癌增殖性方面更准确；PCNA免疫染色反应性较敏感，其类癌诊断的阳性标准应适当提高。LN的表达可预测胃肠道类癌组织的分化、浸润及转移能力。