Ana Tobar

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.

Altered Renal Lipid Metabolism and Renal Lipid Accumulation in Human Diabetic Nephropathy

Animal models link ectopic lipid accumulation to renal dysfunction, but whether this process occurs in the human kidney is uncertain. To this end, we investigated whether altered renal TG and cholesterol metabolism results in lipid accumulation in human diabetic nephropathy (DN). Lipid staining and the expression of lipid metabolism genes were studied in kidney biopsies of patients with diagnosed DN (n = 34), and compared with normal kidneys (n = 12). We observed heavy lipid deposition and increased intracellular lipid droplets. Lipid deposition was associated with dysregulation of lipid metabolism genes. Fatty acid β-oxidation pathways including PPAR-α, carnitine palmitoyltransferase 1, acyl-CoA oxidase, and L-FABP were downregulated. Downregulation of renal lipoprotein lipase, which hydrolyzes circulating TGs, was associated with increased expression of angiopoietin-like protein 4. Cholesterol uptake receptor expression, including LDL receptors, oxidized LDL receptors, and acetylated LDL receptors, was significantly increased, while there was downregulation of genes effecting cholesterol efflux, including ABCA1, ABCG1, and apoE. There was a highly significant correlation between glomerular filtration rate, inflammation, and lipid metabolism genes, supporting a possible role of abnormal lipid metabolism in the pathogenesis of DN. These data suggest that renal lipid metabolism may serve as a target for specific therapies aimed at slowing the progression of glomerulosclerosis.

Embryonic pig pancreatic tissue for the treatment of diabetes in a nonhuman primate model

Xenotransplantation of pig tissues has great potential to overcome the shortage of organ donors. One approach to address the vigorous immune rejection associated with xenotransplants is the use of embryonic precursor tissue, which induces and utilizes host vasculature upon its growth and development. Recently, we showed in mice that embryonic pig pancreatic tissue from embryonic day 42 (E42) exhibits optimal properties as a β cell replacement therapy. We now demonstrate the proof of concept in 2 diabetic Cynomolgus monkeys, followed for 393 and 280 days, respectively. A marked reduction of exogenous insulin requirement was noted by the fourth month after transplantation, reaching complete independence from exogenous insulin during the fifth month after transplantation, with full physiological control of blood glucose levels. The porcine origin of insulin was documented by a radioimmunoassay specific for porcine C-peptide. Furthermore, the growing tissue was found to be predominantly vascularized with host blood vessels, thereby evading hyperacute or acute rejection, which could potentially be mediated by preexisting anti-pig antibodies. Durable graft protection was achieved, and most of the late complications could be attributed to the immunosuppressive protocol. While fine tuning of immune suppression, tissue dose, and implantation techniques are still required, our results demonstrate that porcine E-42 embryonic pancreatic tissue can normalize blood glucose levels in primates. Its long-term proliferative capacity, its revascularization by host endothelium, and its reduced immunogenicity, strongly suggest that this approach could offer an attractive replacement therapy for diabetes.

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.

Tissue expression of squamous cellular carcinoma antigen and Ki67 in hepatocellular carcinoma-correlation with prognosis: A historical prospective study

BackgroundSquamous cellular carcinoma antigen (SCCA) is overexpressed in hepatocellular carcinoma (HCC) tissue and in sera of HCC patients. Our aim was to assess hepatic SCCA immunostaining in a series of HCCs and to correlate its presence with cell proliferation, apoptosis and clinical outcome.MethodsSixty-one HCC patients were included. Liver specimens were obtained either by biopsy (n = 17) or surgically (resection 27, transplantation 17). Immunostaining for AFP, Ki-67, SCCA and TUNEL assay were performed.ResultsSCCA staining was detected in 83.6% of specimens. A statistical significant correlation was found between negative SCCA staining and mortality (p = 0.026) and a higher immunostaining score for Ki67 (p = 0.017). Positive SCCA staining was associated with well and moderate differentiated tumors (p = 0.022). Using multiple logistic regression analysis, Ki67 and TUNEL assay were found to be significant independent predictors of negative SCCA immunostaining. The area under the receiver operator characteristic curve was 0.87. Kaplan-Meier survival analysis revealed a significant difference between the patient group with positive versus negative SCCA immunostaining relating to survival time (p = 0.0106). Cox proportional hazard regression analysis demonstrated that Ki67 immunostaining and liver transplantation or resection were independently associated with mortality.ConclusionsSCCA is overexpressed in HCC. SCCA status is associated with cell proliferation, apoptosis and survival. SCCA and Ki67 staining can predict survival. Our study results support a potential association of negative SCCA expression with other markers of poor outcome in HCC. More studies are needed to clarify the role of SCCA in HCC and expand the knowledge of the SCCA antigen in HCC patients.

Determinants of Long-Term Graft Outcome in Transplant Glomerulopathy

Background. Transplant glomerulopathy (TG) is a renal allograft disease defined by glomerular basement membrane duplication with peritubular capillary basement membrane multilayering (PTCML), and associated with anti-human leukocyte antigen antibodies and C4d. Outcome in TG is poor but variable, and prognostic factors, particularly those affecting long-term outcome, are not well known. We investigated several potentially prognostic clinical and pathologic factors in TG and evaluated estimated glomerular filtration rate (eGFR) slopes to assess graft function and early decline. Methods. We examined all cases of TG from 2001 to 2005 with at least 4-year follow-up after biopsy, excluding those with a second confounding diagnosis. Results. Among 36 cases of pure TG, mean graft age at biopsy was 8.8±6 years. C4d stain was positive in 11 (33%) cases. Clinical characteristics at biopsy were not different based on C4d. C4d was associated with greater PTCML (P=0.03), peritubular capillaritis (P=0.04), and glomerulitis (P=0.03). Death-censored graft survival was significantly associated with interstitial fibrosis (P=0.001), PTCML (P=0.001), and arteriolar hyalinosis (P=0.007), and it showed a trend with proteinuria (P=0.07) and C4d positivity (P=0.08). C4d-positive cases also showed a trend toward rapid graft loss. Analysis of eGFR slopes showed a pattern of preserved, slightly negative slope from transplant until approximately 1 year before biopsy, at which point the slope became significantly more negative (P<0.001). Conclusion. Interstitial fibrosis, PTCML, and arteriolar hyalinosis were significant predictors of graft survival in TG. C4d positivity was associated with a more rapid rate of function decline. eGFR slope data showed significant deterioration in graft function well before the diagnostic biopsy.

Effects of histone deacetylase inhibitors on rat mesangial cells

Glomerular mesangial cells (MCs) proliferate and produce extracellular matrix proteins in many progressive renal diseases. Recently, histone deacetylase inhibitors (HDIs) were shown to have antiproliferative and antifibrogenic effects in some in vitro and in vivo models. Using the [(3)H]-thymidine incorporation test, we have found that the HDI trichostatin A (TSA) effectively inhibits MC growth at nontoxic nanomolar concentrations. Similarly, the HDI valproic acid also inhibited MCs proliferation. Cell-cycle analysis indicated an arrest in G(0)/G(1) phase in response to TSA, which was accompanied by elevation in synthesis of the cyclin-dependent kinase inhibitors (CDKIs) p21/Waf1 and p27/Kip1. TSA treatment suppressed alpha-smooth muscle actin, transforming growth factor-beta1, and collagen protein synthesis by MCs and induced myofibroblast-like appearance of proliferating MCs. In the in vivo model of the anti-Thy1.1-induced glomerulonephritis, TSA and valproic acid treatments significantly suppressed proteinuria. Collectively, these data suggest a therapeutic potential for HDIs in the treatment of mesangial proliferative diseases and glomerulosclerosis.

Acute Phosphate Nephropathy—An Emerging Threat

Background:Acute phosphate nephropathy (APN) is a clinicopathological entity causing renal failure, after ingestion of oral sodium phosphate solution (OSPS). Approximately 25 cases have been described, but OSPS is still widely used. This study reports a further 5 cases and discusses the ever-growing significance of APN. Methods:Five cases of APN were included, 3 retrospectively whereas 2 were diagnosed prospectively. In all, use of OSPS was established, and other causes of nephrocalcinosis were excluded. Results:Average age was 67.4 ± 7.0 years, with a female preponderance (4:1). All patients had hypertension. Baseline serum creatinine: 0.7 to 1.2 mg/dL (creatinine clearance: 52 to 77 mL/min). Time from colonoscopy to presentation was 56 ± 36 days. Serum creatinine levels at presentation: 1.4 to 3.6 mg/dL. Time from colonoscopy to renal biopsy was 123 ± 88 days. Urinalysis showed minimal proteinuria, leucocyturia, and hematuria. One patient had renal glucosuria. All patients were anemic (hemoglobin 8.8–11.4 gr/dL). Serum calcium and phosphate were normal. One required hemodialysis. Mean follow-up was 36 ± 17 months. Serum creatinine levels at end of follow-up were 1.3 to 3.1 mg/dL. Renal function did not recover completely in any patient. Four required long-term erythropoietin treatment. The prominent histopathological findings were calcium–phosphate tubular depositions (100%), interstitial fibrosis (80%), hypertensive changes (80%), and acute tubular degenerative and regenerative changes (60%). Conclusions:APN is a serious, irreversible renal complication of OSPS. It is probably under-recognized. Risk factors include female gender, older age, hypertension, and renal failure, although it may occur with preexisting normal renal function.

Relationship of tumorigenic malignant melanomas to dermal elastin: an expression of tumor/stromal interaction that may be related to prognosis.

Malignant melanomas, which produce a large number of substances active in connective tissue modulation, must contend with the dermis to grow and propagate. We studied the morphologic interactions between tumorigenic malignant melanomas and dermal elastin. Formalin-fixed and paraffin-embedded tissues of 108 tumorigenic malignant melanomas were stained for elastic tissue with the Verhoeff-van Gieson method. Various aspects of the relationship between malignant melanoma and dermal elastin were analyzed in relation to the histologic and clinical data using univariate and multivariate analyses. Tumor thickness, mitotic rate, and the presence of elastin remnants within the tumors were found to be independent negative prognostic factors, the latter with borderline significance. Tumors with more remnants of elastin were associated with higher stage of disease and lymph node and distant metastases. Tumor infiltration between the elastic fibers in the tumor depth was associated with high Clark level, greater tumor thickness, high stage of disease, and lymph node metastases. At least partial preservation of elastic fibers in the tumor depth was a relatively good prognostic factor whereas complete absence of elastin was an adverse factor. Focal or multifocal absence of elastin in the midst of the tumors or in their depth was usually associated with lymphocytic infiltrates. We suggest that tumors with remnants of elastic fibers and/or invasion between elastic fibers in their depth may be fast growing and highly invasive. The absence of elastin within tumors and at their advancing edge may be related to the elaboration of elastin-degrading substances by melanoma cells or various inflammatory cells. Our findings indicate that the relationship between malignant melanomas and dermal connective tissue components, specifically elastin, may have prognostic significance.