Carsten Boltze

High Prognostic Value of p16INK4 Alterations in Gastrointestinal Stromal Tumors

PURPOSE Gastrointestinal stromal tumors (GISTs) represent a distinctive (but histologically heterogeneous) group of neoplasms, the malignant potential of which is often uncertain. To determine the prognostic relevance of p16INK4 alterations in GISTs, we investigated a larger group of GISTs and correlated the genetic findings with clinicopathological factors and patient survival. MATERIAL AND METHODS We evaluated the methylation status of the promotor by methylation-specific polymerase chain reaction (PCR), the presence of mutations by PCR-SSCP-sequencing, the loss of heterozygosity at the p16INK4 locus (using the c5.1 marker), and the immunohistochemical expression of p16INK4 protein in 43 GISTs in 39 patients. RESULTS p16INK4 alterations were found in 25 of 43 GISTs (58.1%), with benign, borderline, or malignant GISTs showing no differences in the type and frequency of alteration. p16INK4 alterations were correlated with a loss of p16INK4 protein expression (P <.01). Patients who had tumors with p16INK4 alterations had a poorer prognosis than patients with tumors without such alterations (P =.02). There was a high predictive value for p16INK4 alterations only in the group of benign and borderline GISTs (P <.01) with regard to clinical outcome. Univariate Coxs proportional hazard regression analysis revealed a strong correlation between p16INK4 alterations, tumor size, mitotic index, and overall survival (P <.02), whereas multivariate Coxs analysis confirmed only p16INK4 alterations as an independent prognostic factor. CONCLUSION We believe that the evaluation of p16INK4 alteration status is a helpful prognosticator, particularly in the benign and borderline groups of GISTs.

5-aza-Cytidine Is a Potent Inhibitor of DNA Methyltransferase 3a and Induces Apoptosis in HCT-116 Colon Cancer Cells via Gadd45- and p53-Dependent Mechanisms

Methyltransferase inhibitors commonly used in clinical trials promote tumor cell death, but their detailed cytotoxic action is not yet fully understood. A deeper knowledge about their apotosis-inducing mechanisms and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b might allow the design of more effective drugs with lower cytotoxicity. 5-aza-cytidine (5-aza-CR), a potent inhibitor of DNMT1, is known to induce demethylation and reactivation of silenced genes. In this study, we investigated the p53 dependence of apoptotic, cell cycle, and growth inhibitory effects of 5-aza-CR, as well as the influence on the expression level of DNMT1, DNMT3a, and DNMT3b in the colon cancer cell line HCT-116. Exposure to 5-aza-CR induced the up-regulation of genes promoting cell cycle arrest and DNA repair (p21WAF1 and GADD45) or apoptosis (p53, RIPK2, Bak1, caspase 5, and caspase 6). In parallel, there was a down-regulation of antiapoptotic Bcl2 protein and the G2/M-mediator cyclin B1. Co-incubation with pifithrin-alpha (PFT-α), a selective p53 inhibitor, restored GADD45, Bcl2, cyclin B1, and p21WAF1 expression levels and almost completely reversed the growth inhibitory, cell cycle, and apoptotic effects of 5-aza-CR. 5-aza-CR treatment caused global demethylation and reactivation of p16INK4 expression. There was a marked decrease in DNMT1 and DNMT3a mRNA expression, with PFT-α reversing these effects. However, 5-aza-CR treatment did not modulate DNMT3b expression. Our data demonstrate that 5-aza-CR action in HCT-116 is mediated by p53 and its downstream effectors p21WAF1 and GADD45. This is the first report to show a link between p53 and regulation of DNMT1 and de novo methyltransferase DNMT3a.

Expression of Matrix Metalloproteinase (MMP)-1, MMP-2, MMP-9, Cathepsin B, and Urokinase Plasminogen Activator in Non–Skull Base Chordoma

We analyzed the expression of proteases and the clinicopathologic significance in non-skull base chordoma (NSBC). By using immunohistochemical techniques, we studied the expression of matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, cathepsin B (CatB), and urokinase plasminogen activator (uPA) in 29 NSBCs and compared these data with clinicopathologic parameters and the expression of cell differentiation markers. Expression of MMP-1 (P = .092), MMP-2 (P = .041), and CatB (P = .058) was associated with nuclear pleomorphism, a previously described adverse prognostic indicator. Expression of cytokeratin 8 correlated with that of MMP-1 (P = .005), MMP-2 (P = .002), and uPA (P = .032). Patients with higher MMP-2 expression had a poorer prognosis than those with lower MMP-2 expression (P = .013). We believe that NSBCs with nuclear pleomorphism or stronger epithelial character have a higher invasive ability than those without. In addition, high MMP-2 expression was an indicator of an unfavorable clinical outcome in NSBC.

Phenotypical and molecular distinctness of sinonasal haemangiopericytoma compared to solitary fibrous tumour of the sinonasal tract

Sinonasal haemangiopericytoma (SN‐HPC) is a rare sinonasal mesenchymal neoplasm of perivascular myoid cell origin. Solitary fibrous tumour (SFT) occurs only very rarely in the sinonasal tract. SFT and soft tissue HPC have been considered a single entity. Recently, recurrent gene fusions involving NAB2‐STAT6 resulting in differential expression of STAT6 were characterized as central molecular events in SFT. However, no data exist for NAB2–STAT6 status or STAT6 expression in SN‐HPC.

Recurrent Mutations within the Amino-Terminal Region of β-Catenin Are Probable Key Molecular Driver Events in Sinonasal Hemangiopericytoma

Sinonasal hemangiopericytoma (SN-HPC) is an uncommon, site-specific, low-grade mesenchymal neoplasm of probable perivascular myoid cell origin. In contrast to solitary fibrous tumors of soft tissue and sinonasal tract origin, SN-HPCs were recently shown to lack recurrent NAB2-STAT6 fusion variants. Other molecular alterations known to occur in some of soft tissue perivascular myoid cell neoplasms were also absent in SN-HPC; thus, the molecular pathogenesis of SN-HPCs remained unknown. Guided by whole-genome sequencing combined with RNA sequencing of an index case, we analyzed a total of six SN-HPCs for mutations within the amino-terminal region of the gene CTNNB1 (cadherin-associated protein), β 1, 88 kDa, encoding β-catenin. All six cases showed missense mutations, with amino acid substitutions clustering at positions 33 to 45, corresponding to the recognition site of the β-catenin destruction complex. Similar CTNNB1 mutations have been described in a variety of epithelial and mesenchymal neoplasms. These mutations prevent β-catenin phosphorylation and proteasomal degradation but promote its nuclear accumulation and subsequent increased transcription of Wingless-related integration site target genes. Consistent with these molecular findings, β-catenin IHC showed consistent diffuse and strong nuclear staining of the tumor cells in all six SN-HPCs. Our results highlight, for the first time, CTNNB1 mutations as the likely initiating molecular events driving SN-HPC tumorigenesis, which places SN-HPC among the growing family of β-catenin-driven mesenchymal neoplasms.

CD97, CD95 and Fas-L clearly discriminate between chronic pancreatitis and pancreatic ductal adenocarcinoma in perioperative evaluation of cryocut sections.

It is a major problem to distinguish between pancreatitis and pancreatic adenocarcinoma when it comes to the perioperative evaluation of pancreatic cryocut sections. In this respect, pathologists are showing a steadily growing interest in the potential application of apoptotic and dedifferentiation factors as diagnostic and prognostic markers. This study investigated the mRNA and protein expression of CD97, CD95 and Fas‐L in snap‐frozen material obtained from human pancreatic ductal adenocarcinomas (PDC; n = 50), tissues from pancreatitis (PT; n = 40) and normal pancreatic tissues (PN; n = 36). Reverse transcription–polymerase chain reaction (RT–PCR) analysis revealed that CD97, CD95 and Fas‐L mRNA were expressed on a similarly high level in all tissues. In contrast, short time immunohistochemical evaluation showed that the CD95 protein was strongly expressed in PT and PN, but not in PDC. Fas‐L protein was expressed strongly in PDC, whereas only weak or no expression was noted in PT or PN. CD97 protein expression was detected only in PT and in poorly differentiated PDC. Our data demonstrate that CD97, CD95 and Fas‐L can be used as additional markers to distinguish between pancreatitis and pancreatic duct cell carcinoma in cryocut sections.

Expression of c-MET, low-molecular-weight cytokeratin, matrix metalloproteinases-1 and -2 in spinal chordoma

Aims:  In skull base chordoma, c‐MET expression has been reported to correlate with younger patient age and favourable prognosis; however, it also contributes to tumour invasiveness, especially in recurrent lesions, suggesting variable roles for c‐MET according to clinical status. The aim of this study was to investigate the significance of c‐MET expression in spinal chordoma, which affects patients who are 10–20 years older than those with skull base chordoma.

Intraperitoneal Versus Intravenous CPT-11 Given Intra- and Postoperatively for Peritoneal Carcinomatosis in a Rat Model

PurposePeritoneal recurrence after resection of colorectal carcinoma is still a major concern. We investigated whether the novel cytostatic drug, CPT-11 (Irinotecan), delivered intraperitoneally (i.p.) and intravenously (i.v.), could inhibit intraperitoneal tumor spread in a rat model.MethodsWe induced intraperitoneal tumor growth using a tumor cell transfer model (106 cells) and divided the rats into the following five groups of eight: group IP1, given CPT-11 i.p. immediately after intraperitoneal tumor cell transfer; group IV1, given CPT-11 i.v. immediately after intraperitoneal tumor cell transfer; group IP2, given CPT-11 i.p. on postoperative days (PODs) 5, 10, and 15; group IV2, given CPT-11 on PODs 5, 10, and 15; and a control group. The rats were killed 30 days after tumor cell transfer, and the tumor weight, number of nodes in the greater omentum and peritoneum, presence of metastases in the liver and lungs, and ascites volume were determined.ResultsCPT-11 inhibited peritoneal tumor growth significantly. The direct intraoperative intraperitoneal application induced a more pronounced effect than the early postoperative intraperitoneal application, but both these application modes were superior to the intravenous route, which had no significant effect.ConclusionCPT-11 was highly efficacious against peritoneal carcinomatosis in this experimental model. The combination of CPT-11 with other cytostatic agents and drugs generating different effector mechanisms may diminish or even prevent intraperitoneal tumor growth.

Expression of the von Hippel-Lindau tumor suppressor gene in nonneoplastic and neoplastic lesions of the thyroid

Alterations of the von Hippel-Lindau (VHL) gene, which is supposed to act as a tumor suppressor gene, can cause hereditary tumors associated with the VHL syndrome and are found in different sporadic cancers as well. While VHL protein is distinctly detectable in thyroid follicles, so far its expression in nonneoplastic and neoplastic lesions of the thyroid has not been investigated comprehensively. To illuminate the role of VHL for thyroid tumorigenesis, we investigated 12 follicular adenomas; 22 follicular carcinomas; 11 papillary carcinomas; 6 poorly differentiated carcinomas (PDTCs); 9 undifferentiated carcinomas (UTCs); 8 medullary carcinomas; 13 cases with nonneoplastic as well as normal thyroid tissue of 10 patients with antibodies against VHL, vascular endothelial growth factor (VEGF); and the proliferation marker MIB1 immunohistochemically; and selected cases by Western blot analysis. VHL was clearly expressed in nonneoplastic lesions and differentiated tumors derived from follicular epithelium, diminished in PDTCs and very weakly or not detectable in UTCs (p=0.001), nonneoplastic, and neoplastic C-cells. Although slightly increased in certain differentiated tumors, VEGF was found to be reduced in UTCs as well. In summary, VHL is expressed differently in nonneoplastic and neoplastic lesions of the thyroid in proportion to the level of differentiation. VHL gene alterations appear to be a late event in tumorigenesis of the thyroid and a reduction in VHL protein expression is associated with a loss of differentiation and increased aggressiveness in thyroid tumors. There is no apparent inverse correlation between VHL and VEGF expression as described for other sporadic carcinomas. Therefore, the role of VHL for angiogenesis and the molecular basis of the inactivation of VHL in thyroid tumors remains to be elucidated.