Brain Metastases from Ovarian Carcinoma: An Evaluation of Prognostic Factors and Treatment.

AIMS AND OBJECTIVES: To review a series of patients with brain metastases from ovarian cancer at a single institution. To describe treatment modalities, their outcomes and to determine prognostic factors. PATIENTS AND METHODS: Between January 1995 and December 2014, 25 patients with ovarian cancer brain metastases were treated at The Sheba Medical Center. The medical records were retrospectively reviewed to collect demographic, clinical, and imaging data as well as the information on the treatment modalities used and their outcomes. RESULTS: Mean patient age at the time of brain metastasis diagnosis was 62.7 years. The median interval between the diagnosis of primary cancer and brain metastasis was 42.3 months. Neurologic deficits, headache, and seizure were the most common symptoms. The brain was the only site of metastasis in 20% of the patients. Active ovarian cancer at the time of diagnosis of brain metastasis was observed in half of the patients with systemic disease. Multiple brain metastases were observed in 25% of the patients. We treated 11 patients with surgery plus radiation therapy protocols in various orders: surgery followed by complementary whole-brain radiation therapy (WBRT), surgery followed by stereotactic radiosurgery (SRS), and surgery followed by WBRT and then by adjuvant SRS. Five patients underwent surgery alone and nine patients were treated with radiation alone (WBRT, SRS, or both). Univariate analysis for predictors of survival demonstrated that age above 62.7 years at the time of central nervous system involvement was a significant risk factor and leptomeningeal disease was a poor prognostic factor in reference to supra-tentorial lesions. Multivariate analysis for predictors of survival, however, showed that multiple brain lesions (>4) were a poor prognostic factor, and multivariate analysis of the time to progression revealed that combined treatments of surgery and radiation resulted in longer median periods of progression-free survival than each modality alone. CONCLUSION: We conclude that the only significant predictors of survival or progression-free survival in our cohort were the number of brain metastases and the treatment modality.

Role of Klotho Protein in Tumor Genesis, Cancer Progression, and Prognosis in Patients with High-Grade Glioma.

BACKGROUND: Klotho, a single-pass transmembrane protein associated with premature aging, acts as a tumor suppressor gene by inhibiting insulin/insulin-like growth factor-1 and fibroblast growth factor pathways. Downregulated Klotho expression is reported in melanoma, mesothelioma, bladder, breast, gastric, cervix, lung, and kidney cancers and is associated with a poor prognosis. Klotho expression and Klotho promoter hypermethylation are predictive factors for patient prognosis. METHODS: To investigate the potential role of Klotho in glioblastoma-multiforme (GBM), 22 GBM samples were collected from the Sheba Tumor Bank and examined. RESULTS: We found that increased Klotho messenger ribonucleic acid (RNA) expression predicted longer survival (P = 0.03) of GBM patients. Methylation analysis was performed on bisulfite-treated deoxyribonucleic acid from the GBM patient samples using ionization time-of-flight mass spectrometry according to the Sequenom EpiTYPER protocols. Klotho promoter hypermethylation was detected in 65% of the GBM samples and correlated significantly with improved survival (P < 0.04). We found 3 major Klotho promotor hypermethylation sites located 585-579 bp, 540-533 bp, and 537-534 bp upstream of the transcription start site. Methylated deoxyribonucleic acid immunoprecipitation studies confirmed these results. Notably, the messenger RNA expression in these GBM samples revealed an unexpected linear correlation with methylation of these 3 hypermethylation sites identified in the Klotho promotor. Thus Klotho expression and methylation could predict prognosis in patients with GBM. CONCLUSIONS: Epigenetic regulation in GBM appears to be complicated. Specific CpG islands affect genes or micro RNAs that interact to control Klotho expression. The diverse effects of these islands may be due to unique factors of GBM.

Antibody array strategy for human growth factor secretome profiling of GH-secreting adenomas.

PURPOSES: To test if the antibody array strategy could be utilized to simultaneously detect the secretion of multiple growth factors by human pituitary GH-adenomas and to measure octreotide-induced alterations. METHODS: Specimens of human pituitary adenomas were cultured and incubated with or without octreotide for 24 h. Conditional media were analyzed by human growth factor antibody array and VEGF concentrations were measured by ELISA. Media were also analyzed for GH concentrations. p21 expression levels were examined by Western blot of the specimens lysates. RESULTS: The antibody arrays successfully identified growth factors secreted by GH-adenomas in vitro. Octreotide treatment induced both elevations and reductions in growth factors secretion. GH response to octreotide was measured, and in this small-sized study resistant and sensitive GH-adenomas presented with no unique secretome pattern of each of the groups. Octreotide-induced VEGF alterations analyzed by the antibody array and by ELISA were not fully matched. CONCLUSIONS: This study suggests that the broad proteomic strategy of antibody arrays may be utilized to study the growth factors secretion pattern of GH-adenomas and its regulation by somatostatin analogs or other compounds.

Radiation-induced vascular malformations in the brain, mimicking tumor in MRI-based treatment response assessment maps (TRAMs).

•Of 310 brain tumors patients recruited, histology of 99 lesions was available.•Of those, 5 were histologically confirmed as radiation-induced malformations.•TRAMs cannot differentiate active tumor from vascular malformation.

Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy.

Cellular distribution and dynamics of mitochondria are regulated by several motor proteins and a microtubule network. In neurons, mitochondrial trafficking is crucial because of high energy needs and calcium ion buffering along axons to synapses during neurotransmission. The trafficking kinesin proteins (TRAKs) are well characterized for their role in lysosomal and mitochondrial trafficking in cells, especially neurons. Using whole exome sequencing, we identified homozygous truncating variants in TRAK1 (NM_001042646:c.287-2A > C), in six lethal encephalopathic patients from three unrelated families. The pathogenic variant results in aberrant splicing and significantly reduced gene expression at the RNA and protein levels. In comparison with normal cells, TRAK1-deficient fibroblasts showed irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration. This study confirms the role of TRAK1 in mitochondrial dynamics and constitutes the first report of this gene in association with a severe neurodevelopmental disorder.

α-methylacyl-CoA racemase (AMACR) expression in chordomas differentiates them from chondrosarcomas.

AIMS: Chordomas and chondrosarcomas are malignant mesenchymal tumours with overlapping morphological and immunohistochemical (IHC) characteristics. Our aim was to evaluate the IHC expression of α-methylacyl-CoA racemase (AMACR/P504S), ß-catenin and E-cadherin in chordomas relative to chondrosarcomas and assess the utility of these markers for differential diagnosis. METHODS: Archival sections of 18 chordomas, 19 chondrosarcomas and 10 mature cartilage samples were immunostained and scored for AMACR, ß-catenin and E-cadherin and the relative differential capacity of each marker was calculated. In addition, AMACR mRNA level was assessed in 5 chordomas by RT-PCR and evaluated by comparative CT method. RESULTS: AMACR and ß-catenin stained 88.9% and 94.1% of the chordomas respectively, 21.1% and 10.5% of the chondrosarcomas correspondingly and none of the mature cartilage samples. E-cadherin stained positively 82.4% of the chordomas, 36.8% of the chondrosarcomas and 42.9% of the mature cartilage cases. Both AMACR and ß-catenin showed statistically significant difference between chordomas and chondrosarcomas (p < 0.001 for both), unlike E-cadherin. AMACR was detected at the mRNA level. CONCLUSIONS: AMACR is expressed in most of the chordomas but only in a minority of chondrosarcomas. AMACR may serve as IHC marker of chordoma with differentiating ability comparable to that of ß-catenin.

Devastating recurrent brain ischemic infarctions and retinal disease in pediatric patients with CD59 deficiency.

Identification of CD59 p.Cys89Tyr mutation in 5 patients from North-African Jewish origin presenting with chronic inflammatory demyelinating polyradiculoneuropathy like disease and chronic hemolysis, led us to reinvestigate an unsolved disease in 2 siblings from the same origin who died 17 years ago. The two patients carried the same CD59 gene mutation previously described by our group. These children had quiet similar disease course but in addition developed devastating recurrent brain infarctions, retinal and optic nerve involvement. Revising the brain autopsy of one of these patients confirmed the finding of multiple brain infarctions of different ages. CD59 protein expression was missing on brain endothelial cells by immunohistochemical staining. This new data expands the clinical spectrum of CD59 mutations and further emphasizes the need for its early detection and treatment.

Human cytomegalovirus viral load in tumor and peripheral blood samples of patients with malignant gliomas.

Malignant gliomas are the most common primary brain tumors in adults. The disease has no known etiology, progresses rapidly, and is fatal despite current therapies. Human cytomegalovirus (HCMV) is a beta herpes virus that is trophic for glial cells and infects 50% to 90% of the adult human population. HCMV-mediated disease in immunosuppressed patients has highlighted the possible role of this virus in the development of other diseases, particularly inflammatory diseases such as vascular diseases, autoimmune diseases, and certain malignancies. Sensitive detection of viral DNA, mRNA, and antigens in tumor tissues, as well as seroepidemiologic evidence, suggest a link between HCMV and several human malignancies. HCMV gene products are proposed to dysregulate multiple cellular pathways involved in oncogenesis, such as cell cycle regulation, apoptosis, migration, and angiogenesis. These theories, currently being researched, suggest that HCMV acts as an oncomodulator in malignancies. We investigated the association between HCMV infection and reactivation, and malignant gliomas. An open, matched case-control, parallel group pilot study was performed in a tertiary referral center. The HCMV viral load in peripheral blood and tumor samples of 19 patients newly diagnosed with glioblastoma multiforme was compared with a matched control cohort comprising 19 patients newly diagnosed with non-malignant brain tumors. There was no significant correlation between peripheral blood and tumor tissue HCMV viral load in patients with glioblastoma multiforme compared to the control cohort. The findings of the present study did not support an oncomodulatory role for HCMV in malignant gliomas.

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.

Steroid-responsive, progressive, focal measles virus brain infection.

Chronic measles virus infection of the brain causes subacute sclerosing panencephalitis (SSPE), a progressive, relentless fatal disorder. We report a 52-year-old male who developed focal, chronic persistent measles virus infection of the brain following interferon and ribavirin therapy for hepatitis C, and who responded to steroid therapy. This case, diametrically different from SSPE, has 2 unique features, its focal nature and its permissive response to steroids, that may add to the understanding of the pathogenesis of SSPE and the mechanism enabling viruses to evade the immune response and establish persistent brain infection.

Brain biopsy in AIDS patients: diagnostic yield and treatment applications.

OBJECTIVE: Central nervous system involvement in AIDS patients can present at any stage of the disease. Brain lesions detected in imaging studies are usually treated empirically. A brain biopsy is indicated in the absence of clinical and radiologic improvement. In the present study, 16 AIDS patients underwent brain biopsy. We evaluated the diagnostic yield of the brain biopsy and the changes in the disease course. MATERIALS AND METHODS: Sixteen consecutive AIDS patients (12 men, 4 women; mean age 40.8 years) underwent a brain biopsy at Sheba Medical Center between 1997 and 2009. A retrospective analysis was performed and the clinical outcome was recorded. RESULTS: Median CD4 count before biopsy was 62.6. Magnetic resonance images revealed multiple lesions in 12 patients and enhancing lesions in 12 patients. A total of 19 biopsies were performed in 16 patients. In the present series, the initial procedures provided a diagnostic yield of 81.25% (13 diagnostic cases from 16 procedures in 16 patients). Two of these patients underwent repeated biopsies that were eventually diagnostic . If repeated biopsies were taken into consideration, the diagnostic yield was 93.75% (15 diagnostic cases in 16 patients). The rate of hemorrhagic complications was 10.5% (2 hemorrhages in 19 procedures).Pathologic examination revealed parasitic and fungal infections in 6 patients (6/16; 38%), progressive multifocal leukoencephalopathy in 4 patients (4/16; 25%), AIDS encephalopathy in 4 patients (4/16; 25%), and lymphoma in 1 patient (1/16; 6%). One patient had a nonspecific inflammatory process (6%). The treatment modality was modified in 12 patients and led to clinical and radiologic improvement in 8 patients. CONCLUSIONS: Brain biopsy should be considered when empiric treatment of central nervous system lesions in AIDS patients fails. Biopsy is diagnostic in the majority of patients. The diagnosis allows for treatment modifications, which lead to clinical and radiologic improvement in some patients.

Long-term response in high-grade optic glioma treated with medically induced hypothyroidism and carboplatin: a case report and review of the literature.

Glioblastoma multiforme (GBM) is the most malignant and frequent brain tumor, with an aggressive growth pattern and poor prognosis despite best treatment modalities. Long-term survival of patients with GBM is rare. Optic glioma represents 0.6-1.2% of all brain tumors. Unlike low-grade optic gliomas in children, optic gliomas in adults are highly aggressive and death usually occurs in less than a year. Prolonged progression-free survival and survival rates have been reported in association with induced hypothyroidism in two clinical trials for recurrent GBM. We present the clinical, radiological, and pathological findings in a patient with inoperable GBM of the optic chiasm. Following failure of initial, standard radiation and temozolomide therapy, chemical hypothyroidism was induced using the antithyroid thioamide, propylthiouracil, followed by carboplatin chemotherapy. Initial thyroid stimulating hormone, free T4, and free T3 analysis was carried out and then monthly. This patient responded rapidly to treatment (clinically and with tumor regression within 4 weeks) on two separate occasions with an extended remission period (2.5 years) and prolonged overall survival (4.5 years). We report the successful long-term tumor response to medically induced chemical hypothyroidism in conjunction with carboplatinum chemotherapy of an adult patient with grade IV GBM of the optic chiasm. These clinical observations find mechanistic support from the recent identification of potent mitogenic actions of the thyroid hormone, L-thyroxine, in malignant glioma through binding to a cognate thyroid hormone receptor on the αvß3 integrin. Approaches to block its activity are now explored in preclinical studies.

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.

Presentation patterns and outcome of gliomatosis cerebri.

A total of 25 patients with gliomatosis cerebri (19 males and 6 females; median age 51 years, range 10-73 years) were diagnosed and treated at the Sheba Medical Center between 1995 and 2009. Of these, 3 patients were 10 years old at the time of diagnosis. Seizures were the initial clinical presentation in 19 patients, focal signs in 16 patients, headaches in 7 patients, cognitive disorder in 4 patients and rapidly progressive hemiparesis in 1 patient. Magnetic resonance imaging (MRI) was performed in the patients and demonstrated a diffuse infiltrative process with a hyperintensity signal on T2-weighted images and a minimal mass effect. Some level of enhancement on MRI was observed in 6 patients. The infiltrative process involved at least two lobes in each patient. Biopsy was performed for diagnosis in the majority of patients. In 1 patient with a markedly rapid deterioration, the diagnosis was established at autopsy. The pathology was compatible with gliomatosis with a diffuse infiltrative low-grade astrocytoma in 21 patients and anaplastic astrocytoma in 5 patients. The patients were treated with whole-brain radiation therapy and 7 patients were treated with combined whole-brain radiation therapy and chemotherapy. Treatment appeared to stabilize 6 patients or improve the clinical condition in 7 patients. Due to the small number of patients in the present study, however, further studies are required to determine the effect of treatment on the natural history of the disease.

Heparanase is highly expressed and regulates proliferation in GH-secreting pituitary tumor cells.

Pituitary tumorigenesis involves remodeling of the extracellular matrix (ECM). Heparanase, an endoglycosidase capable of degrading heparan sulfate, a major polysaccharide constituent of the ECM, is implicated in diverse processes associated with ECM remodeling, such as morphogenesis, angiogenesis, and tumor invasion. The aim of this study was to investigate the possible role of heparanase in pituitary tumorigenesis. Human normal pituitaries and pituitary tumors were examined for heparanase mRNA and protein expression using real-time PCR and immunohistochemistry, respectively. Cell proliferation was assessed by colony formation after heparanase overexpression in GH3 and MtT/S cells. Cell viability and cell cycle progression were evaluated after heparanase gene silencing. Higher heparanase mRNA and protein expression was noted in GH tumors as compared with normal pituitaries. Heparanase overexpression in GH3 and MtT/S cells resulted in a 2- to 3-fold increase in colony number, compared with control cells. Cell viability decreased by 50% after heparanase gene silencing due to induced apoptosis reflected by increased fraction of cleaved poly-ADP-ribose polymerase and sub-G1 events. Notably, exogenously added heparanase enhanced epidermal growth factor receptor, Src, Akt, ERK, and p38 phosphorylation in pituitary tumor cells. Our results indicate that heparanase enhances pituitary cell viability and proliferation and may thus contribute to pituitary tumor development and progression.

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.

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.

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.

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.