Miguel Angel Idoate

Genetic and Epigenetic Modifications of Sox2 Contribute to the Invasive Phenotype of Malignant Gliomas

We undertook this study to understand how the transcription factor Sox2 contributes to the malignant phenotype of glioblastoma multiforme (GBM), the most aggressive primary brain tumor. We initially looked for unbalanced genomic rearrangements in the Sox2 locus in 42 GBM samples and found that Sox2 was amplified in 11.5% and overexpressed in all the samples. These results prompted us to further investigate the mechanisms involved in Sox2 overexpression in GBM. We analyzed the methylation status of the Sox2 promoter because high CpG density promoters are associated with key developmental genes. The Sox2 promoter presented a CpG island that was hypomethylated in all the patient samples when compared to normal cell lines. Treatment of Sox2-negative glioma cell lines with 5-azacitidine resulted in the re-expression of Sox2 and in a change in the methylation status of the Sox2 promoter. We further confirmed these results by analyzing data from GBM cases generated by The Cancer Genome Atlas project. We observed Sox2 overexpression (86%; N = 414), Sox2 gene amplification (8.5%; N = 492), and Sox 2 promoter hypomethylation (100%; N = 258), suggesting the relevance of this factor in the malignant phenotype of GBMs. To further explore the role of Sox2, we performed in vitro analysis with brain tumor stem cells (BTSCs) and established glioma cell lines. Downmodulation of Sox2 in BTSCs resulted in the loss of their self-renewal properties. Surprisingly, ectopic expression of Sox2 in established glioma cells was not sufficient to support self-renewal, suggesting that additional factors are required. Furthermore, we observed that ectopic Sox2 expression was sufficient to induce invasion and migration of glioma cells, and knockdown experiments demonstrated that Sox2 was essential for maintaining these properties. Altogether, our data underscore the importance of a pleiotropic role of Sox2 and suggest that it could be used as a therapeutic target in GBM.

Prognostic value of residual fluorescent tissue in glioblastoma patients after gross total resection in 5-aminolevulinic Acid-guided surgery.

BACKGROUND There is evidence in the literature supporting that fluorescent tissue signal in fluorescence-guided surgery extends farther than tissue highlighted in gadolinium in T1 sequence magnetic resonance imaging (MRI), which is the standard to quantify the extent of resection. OBJECTIVE To study whether the presence of residual fluorescent tissue after surgery carries a different prognosis for glioblastoma (GBM) cases with complete resection confirmed by MRI. METHODS A retrospective review in our center found 118 consecutive patients with high-grade gliomas operated on with the use of fluorescence-guided surgery with 5-aminolevulinic acid. Within that series, the 52 patients with newly diagnosed GBM and complete resection of enhancing tumor (CRET) in early MRI were selected for analysis. We studied the influence of residual fluorescence in the surgical field on overall survival and neurological complication rate. Multivariate analysis included potential relevant factors: age, Karnofsky Performance Scale, O-methylguanine methyltransferase methylation promoter status, tumor eloquent location, preoperative tumor volume, and adjuvant therapy. RESULTS The median overall survival was 27.0 months (confidence interval = 22.4-31.6) in patients with nonresidual fluorescence (n = 25) and 17.5 months (confidence interval = 12.5-22.5) for the group with residual fluorescence (n = 27) (P = .015). The influence of residual fluorescence was maintained in the multivariate analysis with all covariables, hazard ratio = 2.5 (P = .041). The neurological complication rate was 18.5% in patients with nonresidual fluorescence and 8% for the group with residual fluorescence (P = .267). CONCLUSION GBM patients with CRET in early MRI and no fluorescent residual tissue had longer overall survival than patients with CRET and residual fluorescent tissue.

Predicting Metastatic Risk of Gastrointestinal Stromal Tumors: Role of Cell Proliferation and Cell Cycle Regulatory Proteins:

Gastrointestinal stromal tumors (GIST) are a heterogeneous group of neoplasms whose biologic behavior is difficult to predict. The aim of this study is to evaluate the prognostic value in GIST of some oncoproteins involved in regulation of cell proliferation. Tumor size, mitosis, necrosis, and p53, c-myc, and bcl-2 protein expression of 32 GIST were studied. Proliferative index was assessed with Ki67. The 32 cases were grouped into the following clinical categories: (1) clinically benign (BN) were defined as disease-free survival greater than 3 years (n=10); (2) clinically malignant (MN) in which local recurrence or metastasis occurred regardless of the follow-up time (n=1 5); and (3) clinically indeterminate (ID) owing to follow-up <3 years without metastasis or local recurrence (n=seven). Discriminant analysis was used to allocate any tumor to one of the two prognostic groups (BN or MN). In univariate analysis all six factors studied above proved to be of significant prognostic value. Using a multivariate stepwise discriminant analysis to take into account the interrelationship between factors, we found that c-myc expression was the most important prognostic factor, followed, in order of statistical weight, by size and Ki67. These were combined to define a discriminant score ([10.75 x c-myc]+[0.39 x size]+[0.078 x Ki67]-15.54=score), which was capable of correctly identifying tumors in our series whose known clinical behavior was BN or MN in 92% of the cases. The classification score was applied subsequently to the seven clinically ID cases: Three (42.9%) were predicted as BN, and four (57.1%) were predicted as MN. Both expression of oncoprotein c-myc and the proliferative index provide prognostic information in GIST, in addition to morphologically established prognostic factors such as size. These factors in a discriminant analysis proved to be useful for the clinical classification of GIST into BN or MN and to predict the clinical outcome of clinically ID tumors.

Pathological characterization of the glioblastoma border as shown during surgery using 5-aminolevulinic acid-induced fluorescence

Thirty consecutive surgical patients with glioblastoma, were operated upon using fluorescence induced by 5‐aminolevulinic acid as guidance. The fluorescent quality of the tissue was used to take biopsies from the tumor center, from the invasive area around it and from adjacent normal‐looking tissue. These samples were analyzed with HE, Ki‐67 and nestin. Nestin expression in tissue surrounding glioblastoma cases was compared to tissue surrounding vascular lesions, metastasis and hippocampal sclerosis. The rate of gross total resection assessed by volumetric MRI was 83%. Using HE examination as the gold standard, fluorescence identified solid tumor with 100% positive predictive value, invasive areas with 97%, and normal tissue with 67% negative predictive value. Ki67 stained some cells in 69% of the non‐fluorescent samples around the tumor. There was always strong nestin expression around the tumor but it was similar to control cases in non‐glioma lesions with subacute expansion. 5‐aminolevulinic acid fluorescence guidance is very reliable and can help to study the tumor–brain interface. Nestin expression is strong and constant in the tissue around the tumor, but is mostly an acute glial reaction, not specific of the neoplasm. Nestin staining is not recommended as a tumor stem cell marker.

Assessment of Epidermal Growth Factor Receptor and K-Ras Mutation Status in Cytological Stained Smears of Non-Small Cell Lung Cancer Patients: Correlation with Clinical Outcomes

OBJECTIVE Epidermal growth factor receptor (EGFR) and K-ras mutations guide treatment selection in non-small cell lung cancer (NSCLC) patients. Although mutation status is routinely assessed in biopsies, cytological specimens are frequently the only samples available. We determined EGFR and K-ras mutations in cytological samples. METHODS DNA was extracted from 150 consecutive samples, including 120 Papanicolau smears (80%), 10 cell blocks (7%), nine fresh samples (6%), six ThinPrep® tests (4%), and five body cavity fluids (3.3%). Papanicolau smears were analyzed when they had >50% malignant cells. Polymerase chain reaction and direct sequencing of exons 18-21 of EGFR and exon 2 of K-ras were performed. EGFR mutations were simultaneously determined in biopsies and cytological samples from 20 patients. Activity of EGFR tyrosine kinase inhibitors (TKIs) was assessed. RESULTS The cytological diagnosis was adenocarcinoma in 110 samples (73%) and nonadenocarcinoma in 40 (27%) samples. EGFR mutations were identified in 26 samples (17%) and K-ras mutations were identified in 18 (12%) samples. EGFR and K-ras mutations were mutually exclusive. In EGFR-mutated cases, DNA was obtained from stained smears in 24 cases (92%), pleural fluid in one case (4%), and cell block in one case (4%). The response rate to EGFR TKIs in patients harboring mutations was 75%. The mutation status was identical in patients who had both biopsies and cytological samples analyzed. CONCLUSION Assessment of EGFR and K-ras mutations in cytological samples is feasible and comparable with biopsy results, making individualized treatment selection possible for NSCLC patients from whom tumor biopsies are not available.

Expression and serum levels of MMP‐2 and MMP‐9 during human melanoma progression

Matrix metalloproteinases (MMP)‐2 and ‐9 have been implicated in malignant tumour progression, partly because they degrade collagen type IV, a major component of basement membranes. Biopsy specimens from 56 patients with primary melanoma and 7 with cutaneous or nodal metastases were studied by immunohistochemistry. Of 39 patients with estimated good prognosis, 70.5% of melanomas were negative for MMP‐2, compared with only 47% of 17 melanomas in patients who developed metastasis during the 3‐year follow‐up. All skin and nodal metastases were negative for MMP‐2 and positive for MMP‐9. Of 14 thick melanomas, 9 were mostly positive for MMP‐2 expression, suggesting a possible association with the invasiveness of the melanoma. MMP‐2 and MMP‐9 plasma levels were analysed in another 29 patients with melanoma (10 stage I and II, 9 stage III, and 10 stage IV) and in 10 healthy controls. No difference in MMP‐9 plasma levels was found among the groups. Higher MMP‐2 concentrations were observed in patients with metastatic disease (stage IV) than in those with primary melanoma (stage I) or in controls. Serial levels in two patients who passed from stage I to stage III or IV showed no significant difference in MMP‐2 or ‐9 values. We conclude that MMP‐2 expression might be associated with progression of the melanoma. Circulating MMP‐2 and ‐9 levels have shown low sensitivity and specificity, so they do not seem to be good tumour markers in patients with melanoma.

Quantitative volumetric analysis of gliomas with sequential MRI and 11C-methionine PET assessment: patterns of integration in therapy planning

PurposeThe aim of the study was to evaluate the volumetric integration patterns of standard MRI and 11C-methionine positron emission tomography (PET) images in the surgery planning of gliomas and their relationship to the histological grade.MethodsWe studied 23 patients with suspected or previously treated glioma who underwent preoperative 11C-methionine PET because MRI was imprecise in defining the surgical target contour. Images were transferred to the treatment planning system, coregistered and fused (BrainLAB). Tumour delineation was performed by 11C-methionine PET thresholding (vPET) and manual segmentation over MRI (vMRI). A 3-D volumetric study was conducted to evaluate the contribution of each modality to tumour target volume. All cases were surgically treated and histological classification was performed according to WHO grades. Additionally, several biopsy samples were taken according to the results derived either from PET or from MRI and analysed separately.ResultsFifteen patients had high-grade tumours [ten glioblastoma multiforme (GBM) and five anaplastic), whereas eight patients had low-grade tumours. Biopsies from areas with high 11C-methionine uptake without correspondence in MRI showed tumour proliferation, including infiltrative zones, distinguishing them from dysplasia and radionecrosis. Two main PET/MRI integration patterns emerged after analysis of volumetric data: pattern vMRI-in-vPET (11/23) and pattern vPET-in-vMRI (9/23). Besides, a possible third pattern with differences in both directions (vMRI-diff-vPET) could also be observed (3/23). There was a statistically significant association between the tumour classification and integration patterns described above (p < 0.001, κ = 0.72). GBM was associated with pattern vMRI-in-vPET (9/10), low-grade with pattern vPET-in-vMRI (7/8) and anaplastic with pattern vMRI-diff-vPET (3/5).ConclusionThe metabolically active tumour volume observed in 11C-methionine PET differs from the volume of MRI by showing areas of infiltrative tumour and distinguishing from non-tumour lesions. Differences in 11C-methionine PET/MRI integration patterns can be assigned to tumour grades according to the WHO classification. This finding may improve tumour delineation and therapy planning for gliomas.

Cutaneous polyarteritis nodosa

Summary Classic polyarteritis nodosa (PAN) is a segmentary leucocytoclastic vasculitis that affects small‐ and medium‐sized arteries. In 1931, Lindberg (Acta Med Scand 1931; 76: 183–225) described the existence of a cutaneous variant of PAN, without visceral involvement and with a more favourable prognosis. We present four patients diagnosed with cutaneous PAN in our hospital between 1987 and 1998. The study group was composed of three women and one child, whose ages ranged from 11 to 70 years old. The follow‐up period was between 2 and 13 years. Each patient was submitted for an initial clinical, histological and laboratory evaluation and subsequent follow‐up. The presence of nodules was the most frequent cutaneous lesion, preferentially located in the lower limbs. The erythrocyte sedimentation rate was the only parameter that was altered in all patients. Cutaneous biopsies from all patients showed a segmentary leucocytoclastic vasculitis in the arteries of the deep dermis and/or hypodermis. Direct immunofluorescence was positive in just one patient. No visceral involvement was found in any patient. There is confusion about the correct definition of cutaneous PAN. Some clinical findings, such as nodules or livedo reticularis, typical of cutaneous PAN suggest a good prognosis; however, we consider that it is necessary to evaluate these patients for systemic involvement for the possibility of arteritis in other organs as the term polyarteritis suggests.

Angiolymphoid hyperplasia with eosinophilia successfully treated with imiquimod

SIR, Angiolymphoid hyperplasia with eosinophilia (ALHE) is an uncommon, benign vascular proliferation that primarily affects middle-aged women with particular predilection for the head and especially for the periauricular area. Clinically, it is characterized by solitary or multiple red-brown cutaneous papules or nodules, which are often pruritic or painful with little propensity for spontaneous resolution. Numerous therapeutic approaches have been tried for ALHE including destructive techniques such as electrodesiccation and cryotherapy, surgery, radiotherapy, glucocorticoids (topical or intralesional), retinoids, sclerosing injections, pentoxifylline, pulse dye laser, argon laser, interferon-a2a and cytotoxic agents. We report a patient with refractory ALHE who was successfully treated by topical application of imiquimod cream. A 37-year-old white woman was referred to the Department of Dermatology University Clinic of Navarra with an 8-year history of red-brown itchy and tender nodules located on her left ear and preauricular area. The lesion started insidiously with no tendency for spontaneous resolution. There were no regional lymphadenopathy or laboratory abnormalities, especially peripheral eosinophilia. At the time of our first examination, the patient had erythematous papulonodules, tan, dull red in colour, located around the tragus, with involvement of meatus acusticus externus (Fig. 1a). Magnetic resonance imaging showed no arteriovenous malformation. She had previously been treated with excision and with intralesional and topical glucocorticoids, which all provided temporary improvement but no lasting benefits. Histopathological examination confirmed a diagnosis of ALHE. The lesion was composed of small abnormal blood vessels lined by prominent cells with abundant cytoplasm or solid aggregates of epithelioid cells. This abnormal vascular proliferation was disposed around a smallsized artery showing fibrotic change. Mitotic figures were not observed. Surrounding the abnormal vessels was a dense inflammatory infiltrate characterized by lymphocytes intermixed with abundant eosinophils (Fig. 2). The patient started treatment with 5% imiquimod cream five times a week. By

Fas and Fas ligand: expression and soluble circulating levels in cutaneous malignant melanoma.

Summary Background The Fas/Fas ligand (FasL) system plays a key part in maintaining tissue homeostasis via the induction of apoptosis. Functional impairment of the Fas/FasL system is associated with the development and progression of malignancies. Malignant melanoma cells and tissues have been shown to express Fas and FasL to variable extents.