Relationship between serum concentrations of the growth factor pleiotrophin and pleiotrophin-positive tumors

BACKGROUND: Growth factors produced by tumor cells are essential for tumor expansion and may be useful in monitoring tumor progression or therapeutic efficacy if the factors are released into the circulation. In this study, we measured serum levels of pleiotrophin, a secreted heparin-binding growth and angiogenesis factor, in mice bearing human tumor xenografts to determine whether these levels reflected overall tumor burden, and we examined the relationship between tumor expression of pleiotrophin and serum levels of this factor in patients with cancer. METHODS: Pleiotrophin in serum from mice and humans was measured by use of a highly sensitive enzyme-linked immunosorbent assay. For the clinical studies, serum specimens were obtained from 193 patients with various cancers of the gastrointestinal tract and from 28 healthy control subjects. In a subset of 64 cancer patients, serum levels of pleiotrophin were measured at the time of surgery, and tumor expression of this factor was detected immunohistochemically. All P values are two-sided. RESULTS: In mice, serum pleiotrophin levels were found to increase as a function of tumor size. In humans, elevated serum pleiotrophin levels were found in patients with pancreatic cancer (n = 41; P<.0001) and colon cancer (n = 65; P = .0079) but not in patients with stomach cancer (n = 87; P =.42). A statistically significant positive association was found between elevated levels of pleiotrophin in serum drawn at the time of surgery and expression of this factor by tumors (P<.0001). In both mice and humans, serum pleiotrophin levels dropped after successful tumor removal. CONCLUSIONS: Elevated serum pleiotrophin levels can indicate the presence of tumors expressing this factor. Monitoring serum levels of pleiotrophin may prove useful in determining the pharmacologic efficacy of cytotoxic or anti-pleiotrophin therapy.     

Midkine as a novel target gene for the Wilms' tumor suppressor gene (WT1)

Midkine (MK) is a heparin-binding growth factor which is strongly expressed during the midgestation period of mouse embryogenesis. Wilms' tumor is an embryonal kidney malignancy in infants, and WT1 has been identified as its tumor suppressor gene. The high expression level of MK in all Wilms' tumor specimens so far examined and the presence of two WT1 elements (5'-GCGGGGGCG-3') in the human MK promoter region led us to examine the possible role of the WT1 gene product in the regulation of MK gene expression. A gel shift assay verified the complex formation between the WT1 gene product and WT1 consensus sequence of MK gene. DNase1 footprint analysis also demonstrated that the downstream WT1 element was protected from DNase1 cleavage by the addition of the WT1 protein. The human MK promoter fused with the chloramphenicol acetyltransferase gene (phMK2.3kCAT) was co-transfected with an effector plasmid containing the WT1 gene into several cell lines. Transient transfection assays showed suppression of the MK promoter by WT1 co-transfection in recipient cells; deletion of the WT1 binding site abolished the suppression. The evidence reported in this study indicates that MK gene is a newly identified WT1 target gene.     

A longitudinal study of visuospatial discrimination in parkinsonian patients

The heparin-binding growth-associated molecule HB-GAM (also named pleiotrophin) and the retinoic acid-induced heparin-binding protein RIHB (chicken midkine) are developmentally regulated proteins forming a new family of heparin-binding molecules with putative functions during cell growth and differentiation. A direct involvement of these molecules during chondrogenesis in vivo was suggested by their patterns of expression. The putative chondrogenic activity of these molecules was investigated in vitro using micromass cultures from chicken limb bud mesenchymal cells. Exogenous HB-GAM, not RIHB, was found to enhance chondrogenesis in this system. These results provide a strong incentive for considering and further investigating the role of this protein in the control of limb cartilage differentiation.     

Heterogeneous expression of nm23 gene product as a predictor of lymph nodal status in human breast cancer

The nm23 gene was originally identified by differential hybridization of metastatic murine melanoma cell lines. Some experimental studies demonstrated a significantly low metastatic potential of melanoma cell lines transfected with the nm23 gene. In this study, we clarified the relationship between intracellular nm23-immunoreactivity and lymph nodal status of human breast cancer. We analyzed 82 surgically removed breast tumors including 67 invasive carcinomas (ductal, lobular and mucinous carcinomas). The nm23 expression was diffusely positive in the benign tumors and non-invasive carcinomas. Of the invasive ductal carcinomas, lymph node metastasis was found in 67.7% (21/31) of the focally positive/negative cases and in 18.2% (4/22) of the diffusely positive cases (p<0.001). Immunohistochemically, advanced margins of invasive carcinomas with lymph node metastasis were shown to be negative for nm23 expression, while intraductal carcinoma components were positive. This observation suggested that focally positive/negative nm23 expression can be a predictor of lymph node metastasis of invasive ductal breast carcinoma.     

The prevalence of rheumatic diseases in a Filipino urban population: a WHO-ILAR COPCORD Study. World Health Organization. International League of Associations for Rheumatology. Community Oriented Programme for the Control of the Rheumatic Diseases

Recent analysis of bladder tumors has correlated expression of the neurokine midkine (MK) with poor patient survival. To examine a role for MK and the related pleiotrophin (PTN) in tumorigenesis, they were overexpressed in MCF-7 breast carcinoma cells. Expression had no effect on in vitro growth but conferred a growth advantage in vivo. Enhanced tumor growth correlated with increased vascular density and endothelial proliferation, implicating an angiogenic role for MK and PTN. Angiogenic activity of MK and PTN was confirmed in the rabbit corneal assay. Our data therefore identify two novel targets for antiangiogenic drug development.     

Characterization of bovine serum factor triggering the lysis of liposomes via complement activation

Heparin-binding growth factors have been implicated in central nervous system development, regeneration and pathology. To assess the expression pattern and possible function in multiple sclerosis, the heparin-binding growth factors pleiotrophin (PTN), midkine (MK), basic fibroblast growth factor (FGF-2) and one of its receptors (FGFR1/flg) mRNA and protein levels were examined in an experimental autoimmune encephalomyelitis (EAE) model in the Lewis rat. We assessed the time course of expression of PTN, MK and FGF-2 during EAE and determined the cellular origin of FGF-2 and FGFR1 in normal spinal cord and during inflammatory demyelination. Basal expression of PTN and MK mRNAs in normal spinal cords was significantly upregulated after induction of EAE. MK expression was upregulated two to threefold correlating with disease progression, whereas PTN expression reached peak levels threefold above basal levels during the clinical recovery period. FGF-2 mRNA expression was low in normal spinal cord and dramatically increased in correlation with progressive demyelination. FGF-2 was confined to neurons in normal tissue and shifted dramatically to microglia, paralleling their activation during EAE. Double immunohistochemistry revealed colocalization of FGF-2 to activated microglia/macrophages with strongest expression in the macrophage-rich perivascular core area and microglial expression at the edges of white and gray matter perivascular regions. FGFR1, like its ligand, was induced in activated macrophages/microglia. Growth factor expression in demyelinating diseases could serve several functions, e.g., to modulate the activity of microglia/macrophage in an autocrine fashion, to induce the expression of other factors like insulin-like growth factor 1 or plasminogen activator, which can effect regeneration or degeneration, respectively, and finally to stimulate directly localized proliferation and/or regeneration of oligodendrocytes within the lesion area.     

Multiparametric in situ messenger RNA hybridization analysis to detect metastasis-related genes in surgical specimens of human colon carcinomas

We examined the expression of several genes that regulate different steps of metastasis in surgical specimens of human colon carcinomas. The expression of epidermal growth factor receptor (growth), basic fibroblast growth factor [(bFGF), angiogenesis], type IV collagenase (invasion), E-cadherin (adhesion), and multidrug-resistant (mdr)-1 (drug resistance) mRNA was examined using an in situ mRNA hybridization (ISH) technique and Northern blot analysis. Dukes' stage C and D tumors exhibited a higher level of expression (P <0.05) for bFGF, type IV collagenase, and mdr-1 mRNA than Dukes' stage B tumors. The expression level of epidermal growth factor receptor and E-cadherin did not correlate with the stage of the disease. The ISH technique revealed intertumoral heterogeneity for expression of several genes among Dukes' stage B neoplasms. In some Dukes' stage B tumors, we also found intratumoral heterogeneous staining for bFGF and type IV collagenase, with the highest expression level at their invasive edge. In Dukes' stage C and D tumors, the expression of these genes was more uniform. These results recommend the suitability of the multiparametric ISH analysis for metastasis-related genes to identify individual colon cancers with metastatic potential.     

Expression of cyclins D1 and E in human colon adenocarcinomas

Cyclins D1 and E play critical roles in the progression of cells through the G1 phase of the cell cycle. Amplification and/or overexpression of the cyclin D1 gene and aberrant expression of cyclin E have been described in several forms of human cancer. In the present study, we examined the expression of these two genes by Western, Northern and Southern blot analyses in a series of primary human colon carcinomas of various stages and degrees of differentiation and in paired adjacent normal mucosa samples, and also in a series of human colon carcinoma cell lines. About 50% of the colon carcinomas displayed a two to five fold increase in the expression of cyclin D1 mRNA and protein, when compared with the paired normal mucosa samples. Six out of eight carcinomas examined showed a four to nine fold increase in cyclin E mRNA and about 50% of the carcinomas displayed a two to three fold increase in cyclin E protein. Low molecular weight cyclin E-related proteins were observed in four out of ten carcinomas. These changes in cyclins D1 and E occurred in both early and late stage tumors. Three of the six cell lines examined displayed a high expression of cyclin D1 mRNA and protein. A very high level of cyclin E mRNA expression was seen in HCT116 cells and this was associated with the presence of low molecular weight cyclin E-related proteins. None of the primary colon carcinomas nor the six cell lines examined displayed amplification of either the cyclin D1 or cyclin E genes. Thus, an aberrant expression of both cyclins D1 and E occurs in a significant fraction of human colon carcinomas.     

Enzyme-linked immunoassay for midkine, and its application to evaluation of midkine levels in developing mouse brain and sera from patients with hepatocellular carcinomas

Midkine (MK) is a growth factor that promotes neurite outgrowth and survival of neurons, and enhances the plasminogen activator in endothelial cells. A highly sensitive enzyme-linked immunoassay for MK was developed, involving affinity-purified anti-MK antibodies, their biotinylated form, and avidin-beta-galactosidase. The amount of bound avidin-beta-galactosidase was determined using a fluorogenic substrate, 4-methylumbelliferyl-beta-D-galactoside. This method allowed the detection of human and mouse MK in the range of 50 pg-10 ng. Pleiotrophin, which is related to MK in its amino acid sequence, did not show any cross reactivity. Employing this method, the MK levels in the developing mouse brain were determined. The MK level was 2 micrograms/g of wet tissue on the 12th day of gestation, and then steadily decreased during embryogenesis and postnatal development to 30 ng/g two months after birth. The assay method can also be applied to serum samples. Although the MK levels in the sera of normal human subjects were low or undetectable, 0.6-8 ng/ml of MK was detected in samples in the majority of cases of hepatocellular carcinomas.     

Renal pathology in WAGR syndrome

The Wilms' tumor-aniridia-genital anomalies-mental retardation (WAGR) syndrome is associated with an increased risk for developing Wilms' tumor. A right nephrectomy was performed following the diagnosis of Wilms' tumor in a 2-year-old girl with WAGR syndrome and chromosome 11, del 11p13. Pathologic examination revealed intralobar nephrogenic rests and a peripelvic multicystic mass, sharply delineated from the adjacent typical intralobar nephrogenic rests and renal parenchyma, which may represent a cystic Wilms' tumor (cystic partially differentiated nephroblastoma). We studied the expression of the H19 gene by in-situ hybridization performed on paraffin sections of the kidney. H19 is an imprinted maternally-expressed gene that is not translated to protein and functions as a regulatory RNA molecule. It is tightly linked with the paternally-imprinted gene of insulin-like growth factor 2. While IGF2 presumably plays a role in tumorigenesis of Wilms' tumor, H19 is not expressed in the majority of Wilms' tumors. The expression of H19 in the intralobar nephrogenic rests was found to be prominent in the component of the blastema and markedly reduced with differentiation to tubular structures similar to the fetal kidney. The differential diagnosis of hyperplastic intralobar nephrogenic rests from a small Wilms' tumor arising in intralobar nephrogenic rests is difficult. Complete understanding of the chain of molecular events occurring in the evolution of Wilms' tumors may lead to the development of tumor markers to be used on paraffin sections and so help in the differential diagnosis of hyperplasia versus malignant transformation.     

Potential markers (enzymes, proteoglycans) for human liver tumors

In the present work the activities of GGT and G-6-Pase and the content of Cyt P-450 were determined in surgically removed liver specimens (16 hepatocellular carcinomas, 8 focal nodular hyperplasias and 4 adenomas). The activities were compared to the surrounding seemingly normal liver tissue. In the adenomas neither of the enzymes studied showed alterations, characteristic for hepatocarcinogenesis. Four out of 8 FNHs had the enzyme pattern that was found in experimental liver carcinogenesis. Liver carcinoma specimens proved to be heterogenous. Neither elevated GGT nor reduced G-6-Pase activity was consistent in these samples although the average of G-6-Pase activity decreased to 50 percent. Cytochrome P-450 was significantly reduced in the majority of cases, showing the best agreement with the tendency observed in experimental models. As an other approach, the qualitative and quantitative alterations of proteoglycans (PG) were analized in the same tumor samples. The amount of sugar components of PGs the glycosaminoglycans (GAG) increased by many times in liver tumors. Carcinoma samples were characterized by about twentyfold increase in chondroitin sulfate content, compared to normal liver. The enhancement of GAGs is partly the consequence of a selective alteration in PG expression. The amount of perlecan and decorin was found to be increased, while syndecan disappeared from liver carcinomas. These data suggest that malignant transformation in liver is accompanied by specific alteration in the content, composition and structure of PGs. Presumably, these changes have significance in tumor progression and have also the potential to be used as markers for liver tumors.     

In vivo antitumor activity of hexamethylmelamine against human breast, stomach and colon carcinoma xenografts

We have evaluated the antitumor activity of Altretamine (hexamethylmelamine, HMM) on human carcinoma xenografts serially transplanted in nude mice. Five human breast carcinoma xenografts, MX-1, T-61, MCF-7, R-27 and Br-10, were inoculated subcutaneously into female nude mice. Two human stomach carcinoma xenografts, SC-1-NU and St-4, and three human colon carcinoma xenografts, Co-3, Co-4 and Co-6, were inoculated subcutaneously into male nude mice. One pellet of 17 beta-estradiol (0.1 mg/mouse) was inoculated subcutaneously in the mice transplanted with MCF-7 when the tumors were inoculated. HMM was administered per os daily for 4 weeks. MX-1 and T-61 tumors regressed completely after treatment with HMM at a dose of 75 mg/kg (the maximum tolerated dose: MTD) for MX-1 and 25 mg/kg for T-61. Br-10 was sensitive, whereas MCF-7 and R-27 were resistant to HMM at its MTD. HMM exerted the most potent antitumor effect against T-61. Against MX-1, it exerted an antitumor effect equivalent to that of cisplatin or cyclophosphamide. In addition, this agent was effective against all stomach and colon carcinoma xenografts, in particular St-4 (T/C% = 10.7: the mean tumor weight of treated group/the mean tumor weight of control group) and Co-3 (T/C% = 31.5%) which are insensitive to presently available agents. HMM seems worthy of further clinical investigation as a candidate agent to treat breast, stomach, colon and other carcinomas.     

Frequent loss of the P16INK4a gene product in human pituitary tumors

Pituitary tumors develop at a high frequency in retinoblastoma (Rb)-knockout mice; however, defects in the Rb gene are not common in human pituitary tumors. The inverse correlation of Rb and p16 defects in certain human tumors has led us to investigate the expression of p16 in human pituitary tumors as an indirect mechanism of Rb inactivation. By Western blot analysis, the p16 gene product was undetectable in 25 human pituitary tumors, whereas high levels of p16 could be demonstrated in 10 normal human pituitary specimens under the same conditions of protein extraction and immunoblotting. Similar results were obtained at the mRNA level with low to undetectable levels of p16 mRNA in 13 of 14 pituitary tumors relative to 5 normal pituitary specimens. Single-strand conformation polymorphism analysis of p16 exons 1 and 2 revealed no mobility shifts in 25 tumors; however, a quantitative differential PCR analysis revealed diminished amplification of p16 relative to a control gene in 3 of 25 tumors, suggesting homozygous p16 gene loss. We conclude that altered expression of the p16 gene product occurs at a high frequency in human pituitary tumors. This altered expression is not associated with frequent p16 mutation or gene loss, suggesting that alternative mechanisms of gene inactivation and/or altered regulation occur in the majority of these tumors.     

Metastasis-related genes

Genetic changes related to cancer metastasis are overviewed. hst-1/int-2 co-amplification is closely related to the metastatic potential of esophageal carcinomas. Multiple autocrine and paracrine loops including EGF, TGF-alpha/EGF receptor system and HGF/c-met system are related to the biological malignancy of gastric carcinoma in general. On the other hand, K-sam and c-erbB2 amplification are frequently found in the metastatic foci of poorly and well-differentiated type gastric carcinoma. Various splice variants of cell adhesion molecule CD44 are the potent marker of human carcinomas themselves as well as metastases. Reduction in the expression of nm23 is a relatively common event in the metastasis of various human cancers, including stomach and colorectal carcinomas.     

Clinicopathologic features of neuroendocrine carcinomas of the stomach: appraisal of small cell and large cell variants

OBJECTIVE: To clarify the clinicopathologic features of neuroendocrine carcinomas (NECs) of the stomach, we reviewed 56 cases of surgically treated gastric carcinomas with a solid growth or with areas containing patterns characteristic for neuroendocrine (NE) tumors. METHODS: Of the 56 cases reviewed, we selected 33 cases of NEC based both on histologic patterns common to NE tumors and on histochemical/immunohistochemical results. RESULTS: The average age of patients with NEC was 69.8 years (range, 44-92 years). The majority of patients were men (male-female ratio, 23:10). The most frequently affected site was the upper third (46%) of the stomach. Grossly, 9 cases of NEC were fungating and 24 were ulcerated. As compared with 23 patients with non-NEC tumors, the patients with NECs had a worse prognosis. Histologically, NECs had a variety of histologic patterns, including solid, organoid, trabecular, pseudoglandular, spindle cell, and rosettelike. Based on both cell size and morphologic features, we subdivided NECs into 2 variants, namely, small cell NEC and large cell NEC. Our series included 12 cases of small cell NEC and 21 cases of large cell NEC. CONCLUSIONS: Compared with small cell NECs, large cell NECs had a higher mitotic count, larger polygonal cells, a lower nuclear-cytoplasmic ratio, coarser nuclear chromatin, and more frequent conspicuous nucleoli. Large cell NEC was an aggressive tumor with a very poor prognosis (median survival time, 15.2 months; 1-year survival rate, 58%), which approached that for small cell NEC.     

Expression of the Mr 67,000 laminin receptor is an adverse prognostic indicator in human thyroid cancer: an immunohistochemical study

Increased expression of the Mr 67,000 laminin receptor (LR) is a consistent event which appears as cancer cells acquire an invasive and metastatic phenotype. The Mr 67,000 LR is one of the many laminin-binding proteins able to interact with the major glycoprotein of basement membranes, laminin. The recent development of a specific monoclonal antibody directed against the Mr 67,000 LR MLuC5 has allowed us to study large retrospective groups of human cancers with the aim of correlating the Mr 67,000 LR expression to the clinical, pathological, and survival data of the patients. A significant correlation has already been established between the increased expression of Mr 67,000 LR and survival of patients with breast, colon, ovary, lung, and endometrial cancers. In this study, we investigated the possibility that the detection of Mr 67,000 LR in thyroid human cancers could also be of prognostic value. We analyzed the expression of Mr 67,000 LR with immunohistochemistry using MLuC5 antibodies in paraffin sections of 40 benign and 170 malignant thyroid human tumors. We found that Mr 67,000 LR was not usually detectable in normal thyroid tissues adjacent to the lesion. Only 3 of the 40 thyroid adenomas examined (7.5%) presented cells positive for Mr 67,000 LR. For the malignant thyroid tumors examined, we found that 22.3% of papillary thyroid carcinomas, 38% of follicular thyroid carcinomas, 40% of poorly differentiated carcinomas, 25% of medullary carcinomas, and 58.3% of anaplastic carcinomas expressed a high level of Mr 67,000 LR. Although no correlation between the Mr 67,000 LR expression and survival was found in patients with follicular thyroid carcinomas, papillary thyroid carcinomas, anaplastic carcinomas, and medullary carcinomas, there was a significant correlation in primary thyroid cancers. Our data represent the first extensive study of the Mr 67,000 LR expression in human thyroid cancers and strongly suggest that its detection could be of prognostic value in the investigation of primary thyroid cancers.     

Expression of the multidrug resistance-associated protein (MRP) gene in urothelial carcinomas

The intrinsic or acquired resistance of urothelial cancer to chemotherapy is one major obstacle to successful treatment. Generally, the expression level of P-glycoprotein in urothelial cancer is low, so we accordingly investigated the expression of multidrug resistance-associated protein (MRP). We examined the expression of MRP mRNA by means of slot-blotting samples of 11 renal pelvic and/or ureteral tumors, 33 bladder tumors, one lung metastasis from a ureter tumor, 7 non-cancerous urothelia from patients with transitional-cell carcinoma (TCC) and one urothelium from a patient with renal-cell carcinoma (RCC). We also estimated, by Southern blotting, whether or not the MRP gene was amplified in clinical specimens that overexpressed MRP mRNA. MRP was detected immunohistochemically using a polyclonal antibody against MRP. In all, 5 of 11 renal pelvic and/or ureter tumors (45.5%), 17 of 33 bladder tumors (51.5%) and 4 of 7 non-cancerous urothelia of TCC patients (57.1%) expressed more than 2-fold the MRP mRNA levels of drug-sensitive human KB cells. There was no significant difference in the MRP mRNA level between primary and recurrent tumors. Low-grade urothelial carcinomas (G1 and G2 TCCs) expressed significantly higher levels of MRP mRNA than the high-grade G3 TCC. The MRP gene was not amplified in urothelial carcinomas, irrespective of their expression levels of MRP mRNA. Immunohistochemically, MRP was located mainly on the plasma membrane, but also detected on the cytoplasm of cancer cells. MRP may be one mechanism responsible for intrinsic drug resistance in low-grade urothelial cancer.