Hypermethylation-mediated regulation of CD44 gene expression in human neuroblastoma

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination.

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

New trends in the medical management of glioblastoma multiforme: the role of temozolomide chemotherapy.

Standard care for newly diagnosed glioblastoma multiforme (GBM) previously consisted of resection to the greatest extent feasible, followed by radiotherapy. The role of chemotherapy was controversial and its efficacy was marginal at best. Five years ago temozolomide (TMZ) was approved specifically for the treatment of recurrent malignant glioma. The role of TMZ chemotherapy administered alone or as an adjuvant therapy for newly diagnosed GBM has been evaluated in a large randomized trial whose results suggested a significant prolongation of survival following treatment. Findings of correlative molecular studies have indicated that methylguanine methyltransferase promoter methylation may be used as a predictive factor in selecting patients most likely to benefit from such treatment. In this short review the authors summarize the current role of TMZ chemotherapy in the management of GBM, with an emphasis on approved indications and practical aspects.

Deoxyribonucleic acid content as an indicator of progression of squamous cell carcinogenesis in the esophagus: comparative analysis on imprint-cytospin and tissue section preparation.

BACKGROUND: The purpose of this study was to explore the potential use of image analysis on tissue sections preparation as a predictive marker of early malignant changes during squamous cell (SC) carcinogenesis in the esophagus. Results of DNA ploidy quantification on formalin-fixed, paraffin-embedded tissue using two different techniques were compared: imprint-cytospin and 6 microm thick tissue sections preparation. METHODS: This retrospective study included 26 surgical specimens of squamous cell carcinoma (SCC) from patients who underwent surgery alone at the Department of Surgery in CHUV Hospital in Lausanne between January 1993 and December 2000. We analyzed 53 samples of healthy tissue, 43 tumors and 7 lymph node metastases. RESULTS: Diploid DNA histogram patterns were observed in all histologically healthy tissues, either distant or proximal to the lesion. Aneuploidy was observed in 34 (79%) of 43 carcinomas, namely 24 (75%) of 32 early squamous cell carcinomas and 10 (91%) of 11 advanced carcinomas. DNA content was similar in the different tumor stages, whether patients presented with single or multiple synchronous tumors. All lymph node metastases had similar DNA content as their primary tumor. CONCLUSIONS: Early malignant changes in the esophagus are associated with alteration in DNA content, and aneuploidy tends to correlate with progression of invasive SCC. A very good correlation between imprint-cytospin and tissue section analysis was observed. Although each method used here showed advantages and disadvantages; tissue sections preparation provided useful information on aberrant cell-cycle regulation and helped select the optimal treatment for the individual patient along with consideration of other clinical parameters.

Détection des lésions focales hépatiques malignes. Comparaison de l'échographie, de la porto-tomodensitométrie, de la tomodensitométrie tardive et de l'imagerie par résonance magnétique [Detection of malignant focal hepatic lesions. Comparison of ultrasonography, computerized tomography during arterial portography, delayed computerized tomography and magnetic resonance imaging]

AIMS: This study was performed to compare the sensitivity of ultrasonography, computerized tomography during arterial portography, delayed computerized tomography, and magnetic resonance imaging to detect focal liver lesions. Forty three patients with primary or secondary malignant liver lesions were studied prior to surgical intervention. METHODS: The results of the imaging studies were compared with intraoperative examination of the liver, intraoperative ultrasonography and pathology results (29 patients). In the non-operated (14 patients) group, we compared the number of lesions detected by each technique. RESULTS: One hundred and forty six lesions were detected. There was 84% sensitivity with computerized tomography during arterial portography, 61.3% with delayed scan, 63.3% with magnetic resonance imaging and 51% with ultrasonography in operated patients. In patients who did not undergo surgery, magnetic resonance imaging was more sensitive in detecting lesions. CONCLUSIONS: In operated and non-operated patients series, CT during arterial portography had the highest sensitivity, but magnetic resonance imaging had the most consistent overall results.

Interobserver and intraobserver variability of apparent diffusion coefficient measurements in treated malignant hepatic lesions: measurements in the whole lesion versus in the area with the most restricted diffusion

Purpose: The increase of apparent diffusion coefficient (ADC) in treated hepatic malignancies compared to pre-therapeutic values has been interpreted as treatment success; however, the variability of ADC measurements remains unknown. Furthermore, ADC has been usually measured in the whole lesion, while measurements should be probably centered on the area with the most restricted diffusion (MRDA) as it represents potential tumoral residue. Our objective was to compare the inter/intraobserver variability of ADC measurements in the whole lesion and in MRDA. Material and methods: Forty patients previously treated with chemoembolization or radiofrequency were evaluated (20 on 1.5T and 20 on 3.0T). After consensual agreement on the best ADC image, two readers measured the ADC values using separate regions of interest that included the whole lesion and the whole MRDA without exceeding their borders. The same measurements were repeated two weeks later. Spearman test and the Bland-Altman method were used. Results: Interobserver correlation in ADC measurements in the whole lesion and MRDA was as follows: 0.962 and 0.884. Intraobserver correlation was, respectively, 0.992 and 0.979. Interobserver limits of variability (mm2/sec*10-3) were between -0.25/+0.28 in the whole lesion and between -0.51/+0.46 in MRDA. Intraobserver limits of variability were, respectively: -0.25/+0.24 and -0.43/+0.47. Conclusion: We observed a good inter/intraobserver correlation in ADC measurements. Nevertheless, a limited variability does exist, and it should be considered when interpreting ADC values of hepatic malignancies.

Diagnosis and treatment of malignant oddian tumors.

Between 1959 and 1987 we operated on 18 patients for malignant oddian tumor. Eleven had a Whipple resection, 3 a bilio-enteric anastomosis, 4 a local excision with or without bilio-enteric anastomosis. The overall operative mortality was 11% and the median survival was 13.8 months. Three patients are living and without evidence of disease 12, 29 and 30 months, respectively, after a Whipple resection. Because of their anatomy and favourable behaviour, malignant oddian tumors must be separated from the other periampullary tumors. Echography and endoscopic retrograde cholangiopancreatography with deep biopsies are the most efficient diagnostic modalities. With the aim of cure, the treatment is always surgical and relies mainly on duodenopancreatectomy. Those patients with unresectable tumors or unfit for a major procedure should benefit from internal or external biliary drainage. By coexisting duodenal obstruction, a surgical double derivation should be done.

Effect of the TAT-RasGAP(317-326) peptide on apoptosis of human malignant mesothelioma cells and fibroblasts exposed to meso-tetra-hydroxyphenyl-chlorin and light.

BACKGROUND: 5,10,15,20-Tetrakis(m-hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy (PDT) has shown insufficient tumor selectivity for the treatment of pleural mesothelioma. Tumor selectivity of mTHPC-PDT may be enhanced in the presence of the TAT-RasGAP(317-326) peptide which has the potential to specifically sensitize tumor cells to cytostatic agents. MATERIALS AND METHODS: H-meso-1 and human fibroblast cell cultures, respectively, were exposed to two different mTHPC doses followed by light delivery with and without TAT-RasGAP(317-326) administration. mTHPC was added to the cultures at a concentration of 0.04microg/ml and 0.10microg/ml, respectively, 24h before laser light illumination at 652nm (3J/cm(2), 40mW/cm(2)). TAT-RasGAP(317-326) was added to the cultures immediately after light delivery at a concentration of 20microM. The apoptosis rate was determined by scoring the cells displaying pycnotic nuclei. Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: Light delivery associated with 0.04microg/ml mTHPC resulted in a significantly higher apoptosis rate in the presence of TAT-RasGAP(317-326) than without in H-meso-1 cells (p<0.05) but not in fibroblasts. In contrast, 1.0microg/ml mTHPC and light resulted in a significantly higher apoptosis rate in both H-meso-1 cells and fibroblasts as compared to controls (p<0.05) but the addition of TAT-RasGAP(317-326) did not lead to a further significant increase of the apoptosis rate of both H-meso-1 cells and fibroblasts as compared to mTHPC and light delivery alone. CONCLUSION: TAT-RasGAP(317-326) selectively enhanced the effect of mTHPC and light delivery on H-meso-1 cells but not on fibroblasts. However, this effect was mTHPC dose-dependent and occurred only at a low sensitizer dose.

Socioeconomic groups and cancer risk at death in the Swiss Canton of Vaud.

Data collected by the Cancer Registry of the Canton of Vaud, Switzerland, were used to estimate proportional mortality ratios (PMR) and mortality odds ratios (MOR) for various neoplasms according to social class and sector of occupation (agriculture versus others). Mortality ratios were elevated in lower social classes for cancers of the lung (MOR = 1.18 for social class IV or V vs I or II) and other sites strictly related to tobacco (mouth or pharynx, oesophagus and larynx; MOR = 1.70), and (though not significantly) for cancers of the stomach (MOR = 1.16) and uterus (MOR = 1.30 for cervix and 1.47 for corpus uteri). Furthermore, there was a strong negative social class gradient for thyroid cancer (a neoplasm with particularly elevated incidence and mortality in Switzerland), probably attributable to higher prevalence of iodine deficiency in lower social classes (MOR = 3.17). Positive social class gradients emerged for cancers of the intestines (MOR = 0.77 for social class IV or V), skin (MOR = 0.74) and prostate (MOR = 0.87). Agricultural workers showed decreased ratios for cancers of the lung (MOR = 0.75), cervix uteri (MOR = 0.72) and prostate (MOR = 0.80), and excess mortality from cancers of the upper digestive and respiratory sites (MOR = 1.22), stomach (MOR = 1.18), testis (MOR = 2.05) and lympho-haematopoietic neoplasms, particularly myeloma (MOR = 2.14).

Clinicopathological features of eyelid skin tumors. A retrospective study of 5504 cases and review of literature.

Eyelid tumors are the most common neoplasm in daily ophthalmology practice and encompass a wide variety of benign and malignant tumors. In this retrospective study, we report the clinical and histological features of 5504 eyelid skin tumors diagnosed at the Laboratory of Ophthalmopathology of the Hôpital Ophtalmique Jules Gonin, Lausanne, Switzerland, between January 1989 and December 2007. Benign tumors largely predominated over malignant ones, representing 84% of cases in this series, and the 5 most frequent subtypes were squamous cell papilloma (26%), seborrheic keratosis (21%), melanocytic nevus (20%), hidrocystoma (8%), and xanthoma/xanthelasma (6%). Basal cell carcinoma was the most frequent malignant tumor (86%), followed by squamous cell carcinoma (7%) and sebaceous carcinoma (3%). For several tumor subtypes, there was a poor correlation between clinical and histological diagnosis, stressing the numerous pitfalls in the diagnosis of eyelid tumors. We further discuss our results with reference to previously published series.

Malignant gastroduodenal obstruction: palliation with self-expanding metallic stents.

PURPOSE: To evaluate the feasibility, efficacy, and tolerance of self-expanding metallic stent insertion under fluoroscopic guidance for palliation of symptoms related to malignant gastroduodenal obstruction. MATERIALS AND METHODS: Seventy-two patients (38 men, 34 women) aged 25-98 years (mean, 62 years) with duodenal (n = 43), antropyloric (n = 13), surgical gastrojejunostomy (n = 10), or pyloroduodenal (n = 6) malignant obstruction were referred for insertion of self-expanding metallic stents over a 6-year period. Stent insertion was performed with use of a peroral or transgastric approach when necessary (n = 11). RESULTS: Stents were successfully inserted in 70 of the 72 patients (97%) and provided symptom relief in 65 patients (90%). Inserted stents were mainly uncovered vascular (n = 55) or enteral (n = 10) Wallstents. One hundred eight stents were initially inserted: one, two, three, or four stents were indicated in 43, 17, nine, and one patient, respectively. Mean follow-up was 119 days (range, 4-513 days). Mean stent patency was 113 days (range, 4-513 days). Mean survival of patients was 120 days. During follow-up, stent obstruction occurred in seven patients as a result of tumoral overgrowth (n = 5) or ingrowth (n = 2). Complications occurred in 12 of the 72 patients (17%), including stent migration (n = 8), stent fracture (n = 1), duodenal perforation (n = 1), and death related to general anesthesia (n = 1). CONCLUSION: Despite a significant complication rate, self-expanding metallic stent insertion under fluoroscopic guidance appears to be a feasible and useful technique in the palliative management of malignant gastroduodenal obstruction.

Enrichment of human CD4+ V(alpha)24/Vbeta11 invariant NKT cells in intrahepatic malignant tumors.

Invariant NKT cells (iNKT cells) recognize glycolipid Ags via an invariant TCR alpha-chain and play a central role in various immune responses. Although human CD4(+) and CD4(-) iNKT cell subsets both produce Th1 cytokines, the CD4(+) subset displays an enhanced ability to secrete Th2 cytokines and shows regulatory activity. We performed an ex vivo analysis of blood, liver, and tumor iNKT cells from patients with hepatocellular carcinoma and metastases from uveal melanoma or colon carcinoma. Frequencies of Valpha24/Vbeta11 iNKT cells were increased in tumors, especially in patients with hepatocellular carcinoma. The proportions of CD4(+), double negative, and CD8alpha(+) iNKT cell subsets in the blood of patients were similar to those of healthy donors. However, we consistently found that the proportion of CD4(+) iNKT cells increased gradually from blood to liver to tumor. Furthermore, CD4(+) iNKT cell clones generated from healthy donors were functionally distinct from their CD4(-) counterparts, exhibiting higher Th2 cytokine production and lower cytolytic activity. Thus, in the tumor microenvironment the iNKT cell repertoire is modified by the enrichment of CD4(+) iNKT cells, a subset able to generate Th2 cytokines that can inhibit the expansion of tumor Ag-specific CD8(+) T cells. Because CD4(+) iNKT cells appear inefficient in tumor defense and may even favor tumor growth and recurrence, novel iNKT-targeted therapies should restore CD4(-) iNKT cells at the tumor site and specifically induce Th1 cytokine production from all iNKT cell subsets.

Methylation signature of lymph node metastases in breast cancer patients.

BACKGROUND: Invasion and metastasis are two important hallmarks of malignant tumors caused by complex genetic and epigenetic alterations. The present study investigated the contribution of aberrant methylation profiles of cancer related genes, APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14 (ARF), P16 (CDKN2A), P21 (CDKN1A), PTEN, and TIMP3, in the matched axillary lymph node metastasis in comparison to the primary tumor tissue and the adjacent normal tissue from the same breast cancer patients to identify the potential of candidate genes methylation as metastatic markers. METHODS: The quantitative methylation analysis was performed using the SEQUENOM's EpiTYPER? assay which relies on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: The quantitative DNA methylation analysis of the candidate genes showed higher methylation proportion in the primary tumor tissue than that of the matched normal tissue and the differences were significant for the APC, BIN1, BMP6, BRCA1, CST6, ESR-b, P16, PTEN and TIMP3 promoter regions (P<0.05). Among those candidate methylated genes, APC, BMP6, BRCA1 and P16 displayed higher methylation proportion in the matched lymph node metastasis than that found in the normal tissue (P<0.05). The pathway analysis revealed that BMP6, BRCA1 and P16 have a role in prevention of neoplasm metastasis. CONCLUSIONS: The results of the present study showed methylation heterogeneity between primary tumors and metastatic lesion. The contribution of aberrant methylation alterations of BMP6, BRCA1 and P16 genes in lymph node metastasis might provide a further clue to establish useful biomarkers for screening metastasis.

Fibrosarcoma-like lipomatous neoplasm: a reappraisal of so-called spindle cell liposarcoma defining a unique lipomatous tumor unrelated to other liposarcomas

The term "spindle cell liposarcoma" has been applied to liposarcomas (LPSs) composed predominantly or exclusively of spindled cells. These tumors have been considered variants of well-differentiated LPS (WDL), myxoid LPS, and spindle cell lipoma, suggesting that this is a heterogenous group of lesions. Using strict morphologic criteria and molecular and immunohistochemical analyses, we have identified a homogenous group of spindle cell lipomatous tumors, histologically and genetically distinct from other forms of LPS, which we have called "fibrosarcoma-like lipomatous neoplasm." Cases classified as "spindle cell LPS" or "low-grade LPS with spindle cell features" were reviewed. Final selection criteria included: (1) an exclusive low-grade spindle cell component resembling fibrosarcoma; (2) a mixture of bland fibroblastic cells resembling the preadipocyte and early-adipocyte stage of embryonic fat; and (3) molecular-genetic analysis that excluded other forms of lipomatous tumors. Of the initial 25 cases identified, comparative genomic hybridization (CGH) was uninformative in 2 cases; 5 were reclassified as WDL on the basis of molecular data (MDM2 amplification) and 6 as spindle cell lipoma (CGH profiles with a few gains and losses including a constant loss of chromosome 13 and frequent losses of chromosomes 16 and 6). The 12 remaining cases showed flat CGH profiles; of these cases, 11 were negative for DDIT3 gene rearrangements, and 1 result was uninterpretable. Patients ranged in age from 15 to 82 years (mean 50 y); male patients were affected slightly more often (7:5). Tumors arose in the deep (6) and superficial (3) soft tissue of the groin (4), buttock (3), thigh (2), flank (1), shoulder (1), and paratesticular tissue (1) and ranged in size from 2 to 20 cm (mean 7.5 cm). Clinical follow-up in 11 patients (9 mo to 20 y; mean 68 mo) showed no recurrences or metastases. As defined above, "fibrosarcoma-like lipomatous neoplasm" is a unique lipomatous tumor that should be distinguished from WDL/(low-grade) dedifferentiated LPS and myxoid LPS on combined histologic/molecular features because of its better prognosis.

Multifunctional mitoxantrone-conjugated magnetic nanosystem for targeted therapy of folate receptor-overexpressing malignant cells.

BACKGROUND: Targeted delivery of anticancer chemotherapeutics such as mitoxantrone (MTX) can significantly intensify their cytotoxic effects selectively in solid tumors such as breast cancer. In the current study, folic acid (FA)-armed and MTX-conjugated magnetic nanoparticles (MNPs) were engineered for targeted eradication of folate receptor (FR)-positive cancerous cells. Polyethylene glycol (PEG), FA and MTX were covalently conjugated onto the MNPs to engineer the PEGylated FA-MTX-MNPs. The internalization studies were performed using fluorescein isothiocyanate (FITC)-labeled FA-decorated MNPs (FA-FITC-MNPs) in both FR-positive MCF-7 cells and FR-negative A549 cells by means of fluorescence microscopy and flow cytometry. The cellular and molecular impacts of FA-MTX-MNPs were examined using trypan blue cell viability and FITC-labeled annexin V apoptosis assays and 4',6-diamidino-2-phenylindole (DAPI) staining, DNA ladder and quantitative polymerase chain reaction (qPCR) assays. RESULTS: The FR-positive MCF-7 cells showed significant internalization of the FA-FITC-MNPs, but not the FR-negative A549 cells. The FR-positive cells treated with the PEGylated FA-MTX-MNPs exhibited the IC50 values of 3 μg/mL and 1.7 μg/mL, 24 h and 48 h post-treatment, respectively. DAPI staining and DNA ladder assays revealed significant condensation of nucleus and fragmentation of genomic DNA in the FR-positive MCF-7 cells treated with the PEGylated FA-MTX-MNPs as compared to the FR-negative A549 cells. The FITC-labeled annexin V assay confirmed emergence of late apoptosis (>80%) in the FR-positive MCF-7 cells treated with the PEGylated FA-MTX-MNPs, but not in the FR-negative A549 cells. The qPCR analysis confirmed profound cytotoxic impacts via alterations of apoptosis-related genes induced by MTX-FA-MNPs in MCF-7 cells, but not in the A549 cells. CONCLUSION: Our findings evince that the engineered PEGylated FA-MTX-MNPs can be specifically taken up by the FR-positive malignant cells and effectively demolish them through up-regulation of Bcl-2-associated X protein (Bax) and Caspase 9 and down-regulation of AKt. Hence, the engineered nanosystem is proposed for simultaneous targeted imaging and therapy of various cancers overexpressing FRs.