Karla Alvarez

Chromosome 14q loss defines a molecular subtype of clear-cell renal cell carcinoma associated with poor prognosis

Loss of chromosome 14 has been associated with poor outcomes in clear-cell renal cell carcinoma. Expression of HIFα isoforms has been linked to distinct molecular phenotypes of clear-cell renal cell carcinoma. We hypothesized that chromosome 14 loss could lead to a decrease in HIF1α levels, as its gene (HIF1A) resides in this chromosome. We analyzed 112 archival clear-cell renal cell carcinoma tumor specimens with 250K SNP microarrays. We also evaluated expression of HIFα isoforms by qPCR and immunohistochemistry in a subset of 30 patients. Loss of chromosome 14q was associated with high stage (III–IV, P=0.001), high risk for recurrence (P=0.002, RR 2.78 (1.506–5.153)) and with decreased overall survival (P=0.030) in non-metastatic clear-cell renal cell carcinoma. HIF1α mRNA and protein expression was reduced in specimens with loss of 14q (P=0.014) whereas HIF2α was not. Gain of 8q was associated with decreased overall survival (P<0.0001). Our studies confirm an association between 14q loss and clinical outcome in non-metastatic clear-cell renal cell carcinoma patients and that 8q gain is a candidate prognostic marker for decreased overall survival and appears to further decrease survival in patients with 14q loss. We have also identified that differential expression of HIF1α is associated with 14q loss. Further exploration of 8q gain, 14q loss, MYC, HIF1A and EPAS1 (HIF2α) as molecular markers of tumor behavior and prognosis could aid in personalizing medicine for patients with clear-cell renal cell carcinoma.

Virtual-Karyotyping With SNP Microarrays in Morphologically Challenging Renal Cell Neoplasms A Practical and Useful Diagnostic Modality

Approximately 7 % of renal cell tumors are reported to be “unclassified” renal cell carcinoma (RCC) under the current (morphology-based) classification. Genetic lesions characteristic for RCC subtypes can be identified by virtual karyotyping with single nucleotide polymorphisms (SNP) microarrays. In this study, we examined whether virtual karyotypes could be used to better classify a cohort of morphologically challenging/unclassified RCC. Tumor resection specimens from 21 patients were profiled by virtual karyotyping with Affymetrix 10K 2.0 or 250K Nsp SNP mapping arrays and were also evaluated independently by a panel of 7 genito-urinary pathologists. Tumors were classified by the established pattern of genomic imbalances based on a reference cohort of 98 cases with classic morphology and compared with the morphologic diagnosis of the pathologist panel. Virtual karyotyping analysis identified recognized patterns of chromosomal imbalances in all but 1 (16/17 or 94%) cases with successful analysis. Four cases failed owing to low DNA quality. All cases with a panel diagnosis of unclassified RCC and cases in which a majority diagnosis was not reached were classified by their virtual karyotypes. In 1 case, the molecular-based diagnosis was in disagreement with the majority diagnosis. One case with a majority diagnosis of oncocytoma showed a novel genomic pattern not previously identified in the classic morphology cohort. We conclude that virtual karyotypes generated by SNP arrays are a valuable tool for increasing diagnostic accuracy in morphologically challenging or unclassified renal neoplasms. We consider that this technique is a feasible and practical approach for resolving difficult-to-diagnose renal tumors in clinical practice.

Molecular classification of adult renal epithelial neoplasms using microRNA expression and virtual karyotyping.

Oncocytoma, chromophobe renal cell carcinoma (chRCC), and the eosinophilic variant of clear cell RCC (ccRCC) are morphologically similar tumors with significantly different clinical courses. These renal tumor subtypes show characteristic structural genetic changes; however, the mRNA expression patterns of oncocytoma and chRCC are strikingly similar. MicroRNAs (miRNA) are small RNA molecules that regulate the expression of many genes and have been shown to be useful for tumor classification and identification. The miRNA expression was analyzed from formalin-fixed paraffin-embedded tissue in 5 cases each of oncocytoma, ccRCC, papillary RCC, chRCC, and 4 normal kidney tissues using microarrays. Affymetrix single-nucleotide polymorphism arrays were used to detect chromosomal imbalances in each of the tumors. Eighteen miRNAs were significantly different among the 4 tumor types. The microRNA miR-21, a known oncogenic miRNA, was found to be upregulated in papillary and clear cell carcinomas. Four miRNAs could differentiate oncocytomas from chRCCs and the 3 could differentiate papillary RCC from ccRCC, including miR-126, a known vasculogenic miRNA. Of the 18 differentially expressed miRNAs, only 2 correlated with copy number changes in the chromosomal region harboring these genes. One tumor, originally diagnosed as an oncocytoma by morphology, showed a virtual karyotype and miRNA expression pattern consistent with chromophobe carcinoma. Further investigation of the tumor showed vascular invasion. Our study suggests that miRNA expression can be used to differentiate the common subtypes of renal epithelial neoplasms but further validation is necessary. In addition, the lack of correlation between miRNA expression and virtual karyotype suggests a non-copy-number-related mechanism for miRNA gene expression regulation in renal neoplasia.

Reproducibility and performance of virtual karyotyping with SNP microarrays for the detection of chromosomal imbalances in formalin-fixed paraffin-embedded tissues

BackgroundChromosomal imbalances are commonly seen in cancer and inherited genetic diseases. These imbalances may assist in the diagnosis, prognosis, and/or therapeutic management of certain neoplasms. Several methods for detecting chromosomal imbalances, such as, fluorescent in situ hybridization, array comparative genomic hybridization, and single nucleotide polymorphism (SNP) arrays have proven useful in formalin-fixed paraffin-embedded (FFPE) tissues. Here, we report the performance and reproducibility of virtual karyotyping of FFPE tissues with Affymetrix SNP arrays. MethodsVirtual karyotypes from 442 FFPE tumor samples were generated using the Affymetrix GeneChip Mapping 10K Xba 2.0 and/or 250K Nsp SNP mapping arrays. Samples ranged from a few weeks to 17 years in archival storage. Virtual karyotypes were assessed for copy number changes, loss of heterozygosity, and acquired uniparental disomy. ResultsOverall, 75.3% of samples produced interpretable virtual karyotypes with the 10K arrays and 76.7% in the 250K arrays. Parameters for the selection of samples for hybridization were determined, which increased the success rate in both platforms to 81.3 and 92.6%, respectively. FFPE virtual karyotypes generated with both 10K Xba 2.0 and 250K Nsp arrays showed 100% concordance in intralaboratory and interlaboratory reproducibility studies. Samples older than 7 years showed decreased performance. ConclusionsSNP arrays are a reliable, reproducible, and robust platform for the virtual karyotyping of FFPE tumor tissues with performance characteristics adequate for clinical application. Parameters that most significantly affected sample performance were sample age and storage conditions.

Detection of chromosomal aberrations in renal tumors: a comparative study of conventional cytogenetics and virtual karyotyping with single-nucleotide polymorphism microarrays.

CONTEXT -Renal epithelial neoplasms have characteristic chromosomal imbalances, and we have shown previously that virtual karyotypes derived from single-nucleotide polymorphism microarrays can be performed on formalin-fixed, paraffin-embedded tissue. OBJECTIVE -To perform a direct comparison of virtual and conventional karyotypes to evaluate concordance of results. DESIGN -Twenty archival formalin-fixed, paraffin-embedded tumor samples with preexisting, conventional cytogenetic results were analyzed with Affymetrix 10K 2.0 or 250K Nsp single-nucleotide polymorphism microarrays. RESULTS -Nineteen samples yielded adequate virtual karyotypes for interpretation. Eight samples showed complete agreement between the 2 techniques, and 8 samples showed partial agreement. The disease-defining lesions (eg, loss of 3p for clear cell carcinoma) were identified in all 19 cases by virtual karyotypes and in 15 cases by conventional karyotypes. Virtual and conventional karyotypic findings were concordant in the identification of these disease-defining lesions in 86% (13 of 15) of cases. In 3 cases, virtual karyotypes identified lesions consistent with the morphologic diagnosis, whereas the conventional karyotypes were unsuccessful because of insufficient tumor representation or stromal overgrowth. Two cases with acquired uniparental disomy were identified by single-nucleotide polymorphism arrays, and 5 cases with translocations were identified by conventional karyotype. CONCLUSIONS -Our results show that both techniques are able to identify the characteristic chromosomal abnormality for renal tumor subtypes in most cases. Discrepancies can be explained by inherent limitations of each technique, inadequate tumor sampling, and tumor heterogeneity. We conclude that virtual karyotyping is a robust alternative to conventional cytogenetics for the evaluation of chromosomal anomalies in formalin-fixed, paraffin-embedded tissues from renal epithelial neoplasms.

Identification of Tissue of Origin in Body Fluid Specimens Using a Gene Expression Microarray Assay

Body fluid specimens may be the first and only pathologic specimen for clinical evaluation in metastatic cancer cases. The challenge of identifying the tissue of origin in metastatic cancer has led to the emergence of molecular‐based assays, such as the microarray‐based Pathwork Tissue of Origin gene expression test. The ability to use body fluid specimens in this test would be valuable in providing diagnoses to cancer patients without clearly identifiable primary sites. In the current study, the authors evaluated the Tissue of Origin Test for use with malignant effusion specimens.

Oncocytic renal neoplasms: A molecular approach to a common diagnostic challenge

Oncocytic features are a hallmark of renal oncocytoma (OC) but can be seen in other renal tumors such as clear cell renal cell carcinoma (ccRCC) with granular cells and eosinophilic variant of chromophobe RCC (chRCC). Up to 7% of renal neoplasms are ultimately diagnosed as unclassified RCC with oncocytic tumors accounting for a significant number of these. One of the common diagnostic challenges with renal oncocytic tumors is the differentiation between OC and eosinophilic variant of chRCC. The distinction between these 2 entities is critical due to the prognostic implications and patient management decisions. Immunohistochemistry is generally a useful tool for characterizing many renal tumors but is of limited utility in this situation, since staining patterns for these 2 neoplasms are quite similar. However, OC and chRCC have recurrent chromosomal abnormalities that are characteristic of each tumor type. The use of molecular chromosomal analyses as an ancillary tool for this diagnostic challenge has been reported. This case review focuses on the role of molecular analyses, such as virtual karyotyping with SNP arrays, in the diagnosis of unclassified oncocytic renal tumors.

Abstract 3491: Chromosome 14q loss leads to haploinssuficiency of HIF-1a and poor outcomes in metastatic clear cell renal cell carcinoma

Purpose – One of the current key dilemmas in the management of metastatic renal cell carcinoma (mRCC) is the manifestation of innate or acquired resistance to anti-angiogenic therapy. Currently there are no biomarkers of therapeutic efficacy for antiangiogenic agents in clear cell renal cell carcinoma (ccRCC). Loss of chromosomal arm 14q has been previously associated with poor outcomes in surgically treated patients. We evaluated the association between 14q loss and therapeutic resistance in metastatic ccRCC (mRCC) and explored the role of candidate effector genes on this chromosome. Experimental Design – DNA from archival FFPE tumor specimens from 56 mRCC patients treated with sorafenib or bevacizumab was analyzed with Affymetrix 250K Nsp SNP microarrays. We identified the presence of genomic imbalances focusing on the presence of 14q loss. We then evaluated candidate genes in chromosome 14 by qPCR and immunohistochemistry (IHC) in the bevacizumab treated cohort. Results – 14q loss showed a significant association with worse response (SDPD vs. CRPR) in bevacizumab cohort (Fisher9s exact test, p=0.0473), and with shorter progression free survival (PFS) in the sorafenib cohort (p=0.0318). HIF1A mRNA expression was significantly reduced in specimens with loss of 14q and was associated with PFS in the bevacizumab treated cohort (p=0.045). HIF-1alpha protein expression was also reduced in samples with 14q loss. Conclusions – Our studies demonstrate an association between a specific chromosomal alteration (14q loss) and clinical outcome in patients with advanced renal cell carcinoma treated with antiangiogenic agents. Loss of 14q was also associated to changes in the expression of HIF1A. Low HIF1A expression was strongly correlated with shorter PFS. We hypothesize that loss of 14q could lead to an imbalance in HIF-1α/HIF-2α activity, leading to increased HIF-2α, which has been shown to improve tumor cell viability by upregulating various pro-survival pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3491. doi:10.1158/1538-7445.AM2011-3491