Dvora Nass

MicroRNAs accurately identify cancer tissue origin

MicroRNAs (miRNAs) belong to a class of noncoding, regulatory RNAs that is involved in oncogenesis and shows remarkable tissue specificity. Their potential for tumor classification suggests they may be used in identifying the tissue in which cancers of unknown primary origin arose, a major clinical problem. We measured miRNA expression levels in 400 paraffin-embedded and fresh-frozen samples from 22 different tumor tissues and metastases. We used miRNA microarray data of 253 samples to construct a transparent classifier based on 48 miRNAs. Two-thirds of samples were classified with high confidence, with accuracy >90%. In an independent blinded test-set of 83 samples, overall high-confidence accuracy reached 89%. Classification accuracy reached 100% for most tissue classes, including 131 metastatic samples. We further validated the utility of the miRNA biomarkers by quantitative RT-PCR using 65 additional blinded test samples. Our findings demonstrate the effectiveness of miRNAs as biomarkers for tracing the tissue of origin of cancers of unknown primary origin.

MiR‐92b and miR‐9/9* Are Specifically Expressed in Brain Primary Tumors and Can Be Used to Differentiate Primary from Metastatic Brain Tumors

A recurring challenge for brain pathologists is to diagnose whether a brain malignancy is a primary tumor or a metastasis from some other tissue. The accurate diagnosis of brain malignancies is essential for selection of proper treatment. MicroRNAs are a class of small non‐coding RNA species that regulate gene expression; many exhibit tissue‐specific expression and are misregulated in cancer. Using microRNA expression profiling, we found that hsa‐miR‐92b and hsa‐miR‐9/hsa‐miR‐9* are over‐expressed, specifically in brain primary tumors, as compared to primary tumors from other tissues and their metastases to the brain. By considering the expression of only these two microRNAs, it is possible to distinguish between primary and metastatic brain tumors with very high accuracy. These microRNAs thus represent excellent biomarkers for brain primary tumors. Previous reports have found that hsa‐miR‐92b and hsa‐miR‐9/hsa‐miR‐9* are expressed more strongly in developing neurons and brain than in adult brain. Thus, their specific over‐expression in brain primary tumors supports a functional role for these microRNAs or a link between neuronal stem cells and brain tumorigenesis.

Magnetic resonance imaging-guided, high-intensity focused ultrasound for brain tumor therapy

OBJECTIVEMagnetic resonance imaging-guided high-intensity focused ultrasound (MRIgFUS) is a novel technique that may have the potential for precise image-guided thermocoagulation of intracranial lesions. The system delivers small volumetric sonications from an ultrasound phased array transmitter that focuses energy selectively to destroy the target with verification by magnetic resonance imaging-generated thermal maps. A Phase I clinical study was initiated to treat patients with recurrent glioma with MRIgFUS. METHODSTo date, three patients with histologically verified recurrent glioblastoma multiforme have been treated with MRIgFUS. All patients underwent craniectomy 7 to 10 days before therapy to create a bony window for the ultrasound treatment. Sonications were applied to induce thermocoagulation of the enhancing tumor mass. Long-term radiological follow-up and post-treatment tissue specimens were available for all patients. RESULTSMRIgFUS treatment resulted in immediate changes in contrast-enhanced T1-, T2-, and diffusion-weighted magnetic resonance imaging scans in the treated regions with subsequent histological evidence of thermocoagulation. In one patient, heating of brain tissue in the sonication path resulted in a secondary focus outside the target causing neurological deficit. New software modifications were developed to address this problem. CONCLUSIONIn this first clinical report, MRIgFUS was demonstrated to be a potentially effective means of destroying tumor tissue by thermocoagulation, although with an associated morbidity and the inherent invasive nature of the procedure requiring creation of a bone window. A modified technology to allow MRIgFUS treatment through a closed cranium is being developed.

Validation of a microRNA-based qRT-PCR test for accurate identification of tumor tissue origin

Identification of the tissue of origin of a tumor is vital to its management. Previous studies showed tissue-specific expression patterns of microRNA and suggested that microRNA profiling would be useful in addressing this diagnostic challenge. MicroRNAs are well preserved in formalin-fixed, paraffin-embedded (FFPE) samples, further supporting this approach. To develop a standardized assay for identification of the tissue origin of FFPE tumor samples, we used microarray data from 504 tumor samples to select a shortlist of 104 microRNA biomarker candidates. These 104 microRNAs were profiled by proprietary quantitative reverse transcriptase polymerase chain reaction (qRT–PCR) on 356 FFPE tumor samples. A total of 48 microRNAs were chosen from this list of candidates and used to train a classifier. We developed a clinical test for the identification of the tumor tissue of origin based on a standardized protocol and defined the classification criteria. The test measures expression levels of 48 microRNAs by qRT–PCR, and predicts the tissue of origin among 25 possible classes, corresponding to 17 distinct tissues and organs. The biologically motivated classifier combines the predictions generated by a binary decision tree and K-nearest neighbors (KNN). The classifier was validated on an independent, blinded set of 204 FFPE tumor samples, including nearly 100 metastatic tumor samples. The test predictions correctly identified the reference diagnosis in 85% of the cases. In 66% of the cases the two algorithm predictions (tree and KNN) agreed on a single-tissue origin, which was identical to the reference diagnosis in 90% of cases. Thus, a qRT–PCR test based on the expression profile of 48 tissue-specific microRNAs allows accurate identification of the tumor tissue of origin.

Magnetic resonance imaging-guided focused ultrasound for thermal ablation in the brain: a feasibility study in a swine model.

INTRODUCTIONMagnetic resonance imaging (MRI)-guided focused ultrasound is a novel technique that was developed to enable precise, image-guided targeting and destruction of tumors by thermocoagulation. The system, ExAblate2000, is a focused ultrasound delivery system embedded within the MRI bed of a conventional diagnostic MRI scanner. The device delivers small volumetric sonications from an ultrasound phased array transmitter that converge energy to selectively destroy the target. Temperature maps generated by the MRI scanner verify the location and thermal rise as feedback, as well as thermal destruction. To assess the safety, feasibility, and precision of this technology in the brain, we have used the ExAblate system to create predefined thermal lesions in the brains of pigs. METHODSTen pigs underwent bilateral craniectomy to provide a bone window for the ultrasound beams. Seven to 10 days later, the animals were anesthetized and positioned in the ExAblate system. A predefined, 1-cm3 frontal para ventricular region was delineated as the target and treated with multiple sonications. MRI was performed immediately and 1 week after treatment. The animals were then sacrificed and the brains removed for pathological study. The size of individual sonication points and the location of the lesion were compared between the planned dose maps, posttreatment MRI scans, and pathological specimen. RESULTSHigh-energy sonications led to precise coagulation necrosis of the specified targets as shown by subsequent MRI, macroscopic, and histological analysis. The thermal lesions were sharply demarcated from the surrounding brain with no anatomic or histological abnormalities outside the target. CONCLUSIONMRI-guided focused ultrasound proved a precise and an effective means to destroy anatomically predefined brain targets by thermocoagulation with minimal associated edema or damage to adjacent structures. Contrast-enhanced T1-, T2-, and diffusion-weighted MRI scans may be used for real-time assessment of tissue destruction.

Reduced expression of plakoglobin correlates with adverse outcome in patients with neuroblastoma

Plakoglobin and its homologue beta-catenin are cytoplasmic proteins that mediate adhesive functions by interacting with cadherin receptors and signaling activities by interacting with transcription factors. It has been suggested that plakoglobin can suppress tumorigenicity whereas beta-catenin can act as an oncogene. We investigated the correlation between the expression pattern of N-cadherin, beta-catenin, and plakoglobin and tumor behavior in primary tumors of 20 neuroblastoma patients of all stages and in 11 human neuroblastoma cell lines. N-cadherin and beta-catenin were detected in 9 of 11 and 11 of 11 cell lines, respectively, whereas plakoglobin was undetectable or severely reduced in 6 of 11 cell lines. Tumor cells from 16 of 20 patients expressed N-cadherin and 20 of 20 patients expressed beta-catenin at levels similar to those of normal ganglion cells. Plakoglobin was undetectable in 9 of 20 tumors. Plakoglobin deficiency in the primary tumors was significantly associated with adverse clinical outcome. Five of the patients with plakoglobin-negative tumors died whereas four patients are alive without evident disease. In contrast, all patients with plakoglobin-positive tumors are alive; 2 of 11 are alive with the disease and 9 of 11 are alive without evident disease. These results suggest that down-regulation of plakoglobin may be of prognostic value for neuroblastoma patients as predictor of poor outcome.

PURE RHABDOID TUMOR OF THE BLADDER

A 4-year-old girl presented with a single episode of painless macrohematuria. Physical examination was unremarkable. Abdominal ultrasonography and subsequent abdominopelvic computerized tomography demonstrated a localized lesion in the bladder. Cystoscopy revealed a discrete, wide based tumor in the bladder dome, which was confirmed on biopsy as embryonal rhabdomyosarcoma. The patient underwent partial cystectomy, removing the tumor with wide normal margins. Gross pathological examination of the specimen revealed a polypoid mass, measuring 1.8 1.6 2.4 cm., covered by an intact overlying mucosa. On microscopic examination the tumor infiltrated the full thickness of the bladder wall. However, the surgical margins were tumor-free. The specimen demonstrated monomorphous noncohesive large cells with peripherally displaced vesicular nuclei, prominent nucleoli and acidophilic cytoplasm containing typical cytoplasmic inclusion structures. Mitoses were abundant. Positive immunohistochemistry staining for vimentin, smooth muscle actin and epithelial membrane antigen characterized the tumor cells. On the other hand, staining for keratins (MNF116 and CK19), desmin, myoglobin, sarcomeric actin, glial fibrillary acidic protein, synaptophysin, S-100, chromogranin, CD68, MAC-387 and -1 antitripsin were negative. Electron microscopic examination showed the typical aggregates of whorled intermediate filaments that ranged in thickness from 6 to 10 nm. Cross striations, basal lamina and desmosomes were not seen. In view of the typical incision bodies, distinctive immunohistochemistry stainings and the differentiative electron microscopic findings rhabdomyosarcoma was ruled out and rhabdoid tumor was diagnosed. Adjuvant chemotherapy included 8 courses of vincristine, ifosfamide and etoposide followed by 4 courses of vincristine, actinomycin D and cyclophosphamide and 4 courses of vincristine, cyclophosphamide and doxorubicin. At 2-year followup repeat computerized tomography and cystoscopy with the patient under anesthesia demonstrated no evidence of recurrent disease and the patient is continent with a good capacity bladder.

Delayed contrast extravasation MRI: a new paradigm in neuro-oncology

BACKGROUND Conventional magnetic resonance imaging (MRI) is unable to differentiate tumor/nontumor enhancing tissues. We have applied delayed-contrast MRI for calculating high resolution treatment response assessment maps (TRAMs) clearly differentiating tumor/nontumor tissues in brain tumor patients. METHODS One hundred and fifty patients with primary/metastatic tumors were recruited and scanned by delayed-contrast MRI and perfusion MRI. Of those, 47 patients underwent resection during their participation in the study. Region of interest/threshold analysis was performed on the TRAMs and on relative cerebral blood volume maps, and correlation with histology was studied. Relative cerebral blood volume was also assessed by the study neuroradiologist. RESULTS Histological validation confirmed that regions of contrast agent clearance in the TRAMs >1 h post contrast injection represent active tumor, while regions of contrast accumulation represent nontumor tissues with 100% sensitivity and 92% positive predictive value to active tumor. Significant correlation was found between tumor burden in the TRAMs and histology in a subgroup of lesions resected en bloc (r(2) = 0.90, P < .0001). Relative cerebral blood volume yielded sensitivity/positive predictive values of 51%/96% and there was no correlation with tumor burden. The feasibility of applying the TRAMs for differentiating progression from treatment effects, depicting tumor within hemorrhages, and detecting residual tumor postsurgery is demonstrated. CONCLUSIONS The TRAMs present a novel model-independent approach providing efficient separation between tumor/nontumor tissues by adding a short MRI scan >1 h post contrast injection. The methodology uses robust acquisition sequences, providing high resolution and easy to interpret maps with minimal sensitivity to susceptibility artifacts. The presented results provide histological validation of the TRAMs and demonstrate their potential contribution to the management of brain tumor patients.

Delayed Contrast Extravasation MRI for Depicting Tumor and Non-Tumoral Tissues in Primary and Metastatic Brain Tumors

The current standard of care for newly diagnosed glioblastoma multiforme (GBM) is resection followed by radiotherapy with concomitant and adjuvant temozolomide. Recent studies suggest that nearly half of the patients with early radiological deterioration post treatment do not suffer from tumor recurrence but from pseudoprogression. Similarly, a significant number of patients with brain metastases suffer from radiation necrosis following radiation treatments. Conventional MRI is currently unable to differentiate tumor progression from treatment-induced effects. The ability to clearly differentiate tumor from non-tumoral tissues is crucial for appropriate patient management. Ten patients with primary brain tumors and 10 patients with brain metastases were scanned by delayed contrast extravasation MRI prior to surgery. Enhancement subtraction maps calculated from high resolution MR images acquired up to 75 min after contrast administration were used for obtaining stereotactic biopsies. Histological assessment was then compared with the pre-surgical calculated maps. In addition, the application of our maps for prediction of progression was studied in a small cohort of 13 newly diagnosed GBM patients undergoing standard chemoradiation and followed up to 19.7 months post therapy. The maps showed two primary enhancement populations: the slow population where contrast clearance from the tissue was slower than contrast accumulation and the fast population where clearance was faster than accumulation. Comparison with histology confirmed the fast population to consist of morphologically active tumor and the slow population to consist of non-tumoral tissues. Our maps demonstrated significant correlation with perfusion-weighted MR data acquired simultaneously, although contradicting examples were shown. Preliminary results suggest that early changes in the fast volumes may serve as a predictor for time to progression. These preliminary results suggest that our high resolution MRI-based delayed enhancement subtraction maps may be applied for clear depiction of tumor and non-tumoral tissues in patients with primary brain tumors and patients with brain metastases.

Epidemiology of Gliomas in Israel: A Nationwide Study

Background: Glial brain tumors span a wide range of neoplasms with distinct clinical and histopathological features. This report presents the descriptive epidemiology of glial tumors by histological subtype and tumor behavior. Methods: The study population included all incident cases of glial tumors diagnosed in Israel during March 2001 to July 2003. Age-standardized incidence rates (ASR) were calculated using the world population as a standard. Results: A total of 548 tumors were diagnosed, of which 520 had histological confirmation. The ASR of all adult (>20 years) glial tumors was 5.82/100,000 (7.11 for males; 4.75 for females, p < 0.001). The majority of tumors (78%) were classified as high grade; astrocytic tumors were the most frequent (85%), with glioblastoma multiforme accounting for 70% of them. A significant positive association was shown between age at diagnosis and grade. The highest ASR was seen for Europe- and-American-born, followed by Israeli, Asian and African-born individuals (6.78, 5.86, 4.94 and 3.84/100,000, respectively). Conclusions: In general, these results describing data of incident cases of pathologically validated glial tumors are consistent with previous reports. To enhance our understanding of these diseases, epidemiological studies should rely on well-defined histological tumor types, incorporating comprehensive information which will allow comparability between different groups of patients.