Design and Reporting Considerations for Genetic Screening Tests.

Testing asymptomatic individuals for unsuspected conditions is not new to the medical and public health communities. Protocols to develop screening tests are well established. However, the application of screening principles to inherited diseases presents unique challenges. Unlike most screening tests, the natural history and disease prevalence of most rare inherited diseases in an unselected population are unknown. It is difficult or impossible to obtain a truth set cohort for clinical validation studies. As a result, it is not possible to accurately calculate clinical positive and negative predictive values for likely pathogenic variants, which are commonly returned in genetic screening assays. In addition, many of the genetic conditions included in screening panels do not have clinical confirmatory tests. All these elements are typically required to justify the development of a screening test, according to the World Health Organization screening principles. Nevertheless, as the cost of DNA sequencing continues to fall, more individuals are opting to undergo genomic testing in the absence of a clinical indication. Despite the challenges, reasonable estimates can be deduced and used to inform test design strategies. Herein, we review basic test design principles and apply them to genetic screening.

Evaluation for Genetic Disorders in the Absence of a Clinical Indication for Testing: Elective Genomic Testing.

The increasing quality and diminishing cost of next-generation sequencing has transformed our ability to interrogate large quantities of genetic information. This has led to a dramatic increase in the number of elective genomic tests performed. In this article, elective test denotes a test that a patient chooses to undertake without a clinical indication. The variety of elective genomic testing options is considerable. Because these offerings provide differing levels of sensitivity and specificity, it can be difficult to choose among them. A simple rubric to compare offerings is not readily available. We propose a framework designated completeness that evaluates both analytical and interpretative components of genomic tests. We then illustrate how this framework can be used to evaluate the expanding landscape of elective genomic testing.

The Spectrum of Clinical Utilities in Molecular Pathology Testing Procedures for Inherited Conditions and Cancer: A Report of the Association for Molecular Pathology.

Clinical utility describes the benefits of each laboratory test for that patient. Many stakeholders have adopted narrow definitions for the clinical utility of molecular testing as applied to targeted pharmacotherapy in oncology, regardless of the population tested or the purpose of the testing. This definition does not address all of the important applications of molecular diagnostic testing. Definitions consistent with a patient-centered approach emphasize and recognize that a clinical test result's utility depends on the context in which it is used and are particularly relevant to molecular diagnostic testing because of the nature of the information they provide. Debates surrounding levels and types of evidence needed to properly evaluate the clinical value of molecular diagnostics are increasingly important because the growing body of knowledge, stemming from the increase of genomic medicine, provides many new opportunities for molecular testing to improve health care. We address the challenges in defining the clinical utility of molecular diagnostics for inherited diseases or cancer and provide assessment recommendations. Starting with a modified analytic validity, clinical validity, clinical utility, and ethical, legal, and social implications model for addressing clinical utility of molecular diagnostics with a variety of testing purposes, we recommend promotion of patient-centered definitions of clinical utility that appropriately recognize the valuable contribution of molecular diagnostic testing to improve patient care.

A multicenter, cross-platform clinical validation study of cancer cytogenomic arrays.

Cytogenomic microarray analysis (CMA) offers high resolution, genome-wide copy number information and is widely used in clinical laboratories for diagnosis of constitutional abnormalities. The Cancer Genomics Consortium (CGC) conducted a multiplatform, multicenter clinical validation project to compare the reliability and inter- and intralaboratory reproducibility of this technology for clinical oncology applications. Four specimen types were processed on three different microarray platforms-from Affymetrix, Agilent, and Illumina. Each microarray platform was employed at two independent test sites. The results were compared in a blinded manner with current standard methods, including karyotype, FISH, or morphology. Twenty-nine chronic lymphocytic leukemia blood, 34 myelodysplastic syndrome bone marrow, and 30 fresh frozen renal epithelial tumor samples were assessed by all six laboratories. Thirty formalin fixed paraffin embedded renal tumor samples were analyzed at the Affymetrix and Agilent test sites only. All study samples were initial diagnostic samples. Array data were analyzed at each participating site and were submitted to caArray for central analysis. Laboratory interpretive results were submitted to the central analysis team for comparison with the standard-of-care assays and for calculation of intraplatform reproducibility and cross-platform concordance. The results demonstrated that the three microarray platforms 1) detect clinically actionable genomic changes in cancer compatible to standard-of-care methods; 2) further define cytogenetic aberrations; 3) identify submicroscopic alterations and loss of heterozygosity (LOH); and 4) yield consistent results within and between laboratories. Based on this study, the CGC concludes that CMA is a sensitive and reliable technique for copy number and LOH assessment that may be used for clinical oncology genomic analysis.

Oncocytoma-like renal tumor with transformation toward high-grade oncocytic carcinoma: a unique case with morphologic, immunohistochemical, and genomic characterization.

Renal oncocytoma is a benign tumor with characteristic histologic findings. We describe an oncocytoma-like renal tumor with progression to high-grade oncocytic carcinoma and metastasis. A 74-year-old man with no family history of cancer presented with hematuria. Computed tomography showed an 11 cm heterogeneous multilobulated mass in the right kidney lower pole, enlarged aortocaval lymph nodes, and multiple lung nodules. In the nephrectomy specimen, approximately one third of the renal tumor histologically showed regions classic for benign oncocytoma transitioning to regions of high-grade carcinoma without sharp demarcation. With extensive genomic investigation using single nucleotide polymorphism-based array virtual karyotyping, multiregion sequencing, and expression array analysis, we were able to show a common lineage between the benign oncocytoma and high-grade oncocytic carcinoma regions in the tumor. We were also able to show karyotypic differences underlying this progression. The benign oncocytoma showed no chromosomal aberrations, whereas the high-grade oncocytic carcinoma showed loss of the 17p region housing FLCN (folliculin [Birt-Hogg-Dubé protein]), loss of 8p, and gain of 8q. Gene expression patterns supported dysregulation and activation of phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (Akt), mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK), and mechanistic target of rapamycin (serine/threonine kinase) (mTOR) pathways in the high-grade oncocytic carcinoma regions. This was partly attributable to FLCN underexpression but further accentuated by overexpression of numerous genes on 8q. In the high-grade oncocytic carcinoma region, vascular endothelial growth factor A along with metalloproteinases matrix metallopeptidase 9 and matrix metallopeptidase 12 were overexpressed, facilitating angiogenesis and invasiveness. Genetic molecular testing provided evidence for the development of an aggressive oncocytic carcinoma from an oncocytoma, leading to aggressive targeted treatment but eventual death 39 months after the diagnosis.

Gliosarcoma arising from an oligodendroglioma (oligosarcoma).

Gliosarcoma, a biphasic tumor with both mesenchymal and glial elements, is typically considered a variant of astrocytoma (glioblastoma), WHO Grade IV. A 57-year-old man presented with altered mental status and was found to have a large right frontal mass. Biopsy and subsequent subtotal resection revealed a WHO Grade II oligodendroglioma with classic histological features, expression of IDH1 R132H mutant protein, and chromosome 1p19q co-deletion. Fifteen months later, the patient developed recurrent tumor composed of intersecting fascicles of spindled cells with necrosis and a high mitotic index. The recurrent tumor stained for both mesenchymal and glial elements, consistent with the diagnosis of gliosarcoma, and showed retained IDH1 R132H expression. By FISH analysis, the gliosarcoma showed no evidence of 1p19q co-deletion. We performed SNP arrays and detailed SNP analysis of both the oligodendroglioma and the gliosarcoma. This demonstrated loss of heterozygosity (LOH) of chromosomes 1 and 19 in the gliosarcoma with retention of the same full-length chromosomes 1 and 19 found intact in the oligodendroglioma. Not surprisingly, the gliosarcoma harbored multiple additional alterations, consistent with clonal evolution. There have been only rare reports of sarcomatous transformation of oligodendroglioma ("oligosarcoma") and most were published prior to the development of modern genetic modalities. Here we present a case with detailed genetic evidence that suggests that mesenchymal metaplasia sarcomatous transformation is possible in classic oligodendrogliomas with 1p19q codeletions.

A common 8q (MYC) amplification detected in a multifocal anaplastic astrocytoma by SNP array karyotyping.

The distinction of multifocal versus multicentric gliomas can conceivably have important therapeutic implications. We present a 27-year-old man with two radiologically distinct non-enhancing infiltrative masses in the anterior frontal lobe and the posterior temporoparietal region. No intervening disease was evident on MRI modalities; the lesions were stable over a period of many months. He underwent two separate resections a few months apart. Given the question of whether his tumors represented two de novo primary multicentric tumors or one multifocal tumor, single nucleotide polymorphism (SNP) array karyotyping and in situ hybridization studies were performed on both tumors. The two tumor profiles looked remarkably similar, histologically and genetically: both were anaplastic astrocytomas with a common 33Mb gain/ amplification of 8q23.3-q24.3, including MYC amplification, suggesting a monoclonal origin. The temporoparietal neoplasm showed several additional genetic alterations. This case illustrates that even with today's advanced neuroimaging modalities, extensive radiologically invisible tumor may be present between seemingly separate sites of glioma involvement. Thus modern global genomic studies of such tumors may help distinguish whether multiple tumors represent one extensive neoplasm with microscopically invasive disease or multiple genetically distinct tumors.

Interplay among BRAF, p16, p53, and MIB1 in pediatric low-grade gliomas.

BRAF rearrangements and BRAF V600E point mutations are recurring events in pediatric low-grade gliomas. However, their clinical significance, including possible interactions between these markers and other glioma biomarkers, is unclear. In this study a retrospective cohort of 198 pediatric low-grade gliomas (including 40 treated with adjuvant therapy) was analyzed for BRAF rearrangements, BRAF V600E, p16/CDKN2A deletion, p53 expression, and MIB1 proliferation index. In tumors with BRAF rearrangement, homozygous p16 deletion correlated with shorter progression-free survival (P = .04). A high MIB1 proliferation index trended toward worse response to adjuvant radiotherapy compared to BRAF-rearranged, p16-intact tumors (P = .08). On multivariate analysis, the 2 most consistently powerful independent adverse prognostic markers were midline location (P = .0001) and p16 deletion (P = .03). Tumors with BRAF V600E had a strong trend toward an increased risk for progression (hazard ratio = 2.48, P = .07), whereas those with BRAF rearrangement had a milder trend toward reduced risk (hazard ratio = .54, P = .15). These data suggest that p16 deletion adversely impacts the outcomes of BRAF-driven gliomas, that high proliferation index may be a better marker of progression risk than BRAF, that BRAF rearrangement and BRAF V600E might not necessarily produce comparable outcomes, and that none of these markers is stronger than tumor location in determining prognosis in pediatric low-grade gliomas.

Lynch syndrome-associated extracolonic tumors are rare in two extended families with the same EPCAM deletion.

OBJECTIVES: The Lynch syndrome (LS) is an inherited cancer syndrome showing a preponderance of colorectal cancer (CRC) in context with endometrial cancer and several other extracolonic cancers, which is due to pathogenic mutations in the mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2. Some families were found to show a LS phenotype without an identified MMR mutation, although there was microsatellite instability and absence of MSH2 expression by immunohistochemistry. Studies of a subset of these families found a deletion at the 3' end of the epithelial cell adhesion molecule (EPCAM) gene, causing transcription read-through resulting in silencing of MSH2 through hypermethylation of its promoter. The tumor spectrum of such families appears to differ from classical LS. METHODS: Our study of two large families (USA Family R and Dutch Family A) with an EPCAM deletion was carried out using each institution's standard family study protocol. DNA was extracted from peripheral blood and EPCAM deletion analysis was performed. RESULTS: Both families were found to harbor the same deletion at the 3' end of EPCAM. Analysis showed that the deletion originated from a common ancestor. Family R and Family A members showed segregation of CRC with the presence of this EPCAM mutation. Compared with classic LS, there were almost no extracolonic cancers. CONCLUSIONS: Members of Family R and Family A, all with the same EPCAM deletion, predominantly presented with CRC but no LS-associated endometrial cancer, confirming findings seen in other, smaller, LS families with EPCAM mutations. In these EPCAM mutation carriers, cancer surveillance should be focused on CRC.

Clinical genomics of renal epithelial tumors.

Kidney and upper urinary tract cancers account for approximately 54,000 cases every year in the United States, and represent about 3.7% of adult malignancies, with more than 13,000 annual deaths. Classification of renal tumors is typically based on histomorphologic characteristics but, on occasion, morphologic characteristics are not sufficient. Each of the most common histologic subtypes harbors specific recurrent genetic abnormalities, such as deletion of 3p in conventional clear cell carcinoma, trisomy 7 and 17 in papillary renal cell carcinoma, multiple monosomies in chromophobe renal cell carcinoma, and a nearly diploid genome in benign oncocytomas. Knowledge of this information can provide diagnostic support and prognostic refinement in renal epithelial tumors. Identification of the specific subtype of a renal tumor is critical in guiding surveillance for recurrence and the appropriate use of targeted therapies. Cytogenomic arrays are increasingly being used as a clinical tool for genome-wide assessment of copy number and loss of heterozygosity in renal tumors. In addition, the improved understanding of the hereditary causes of renal tumors and their role in sporadic malignancies has led to the development of more effective targeted therapies. This review summarizes the genetic and genomic changes in the most common types of renal epithelial tumors and highlights the clinical implications of these aberrations.

Sarcoma arising as a distinct nodule within glioblastoma: a morphological and molecular perspective on gliosarcoma.

Gliosarcoma is a variant of glioblastoma and is characterized by distinct glial and sarcomatous components. Typically, there is no macroscopic boundary between the components and special stains are often required to distinguish the glial and sarcomatous elements. Some studies suggest similar genetic alterations in both components pointing to a common origin. We present an extreme case of gliosarcoma arising as a discrete fibrous nodule adjacent to a typical glioblastoma. A 65 year-old woman presented with progressive weakness, seizures and right-sided hemiparesis. CT scan demonstrated an irregular enhancing left frontal lobe mass and an adjacent discrete nodule with different imaging characteristics. The unique nature of this macroscopically biphasic neoplasm allowed us to compare the molecular characteristics of glial and sarcomatous elements which were strikingly similar except for small losses and gains in Chr 3. Studies are under way to determine the significance of chromosome 3 alterations in gliosarcomas.

Assessment of HER2 gene status in breast carcinomas with polysomy of chromosome 17.

BACKGROUND: The current study was performed to determine the impact of polysomy 17 on the interpretation of HER2 testing of invasive breast carcinomas using fluorescent in situ hybridization methods. Current American Society of Clinical Oncology/College of American Pathologists guidelines define HER2-positive tumors as those with >6 HER2 genes per nucleus or those with HER2/CEP17 (chromosome 17) ratio >2.2. These guidelines are potentially contradictory in tumors with polysomy of chromosome 17. METHODS: Seventy-two breast carcinoma cases with reported polysomy of chromosome 17 (≥ 3 CEP17 signals on average) by fluorescent in situ hybridization were identified, and the corresponding HER2 immunohistochemistry was obtained. The HER2 status of the archived samples was reviewed, and the tumors were recategorized according to the 2007 American Society of Clinical Oncology/College of American Pathologists guidelines. RESULTS: The average CEP17 copy number for the group was 4.5 (range, 3.0-10.4). Thirty-three (45.8%) cases had >6 copies of HER2 per nucleus. Twenty-one cases (29.2%) qualified as HER2 gene amplified using the HER2/CEP17 ratio (>2.2) guideline. All these cases had >6 HER2 signals, which represented 63.6% of all cases with >6 HER2 signals. HER2 protein expression showed significant positive correlations with both HER2 gene copy number and HER2/CEP17 ratio (P < .01, r(s) = 0.56 and 0.64, respectively). CONCLUSIONS: Increased CEP17 signals detected in invasive breast carcinomas may lead to discordant interpretation of gene amplification in a significant proportion of the cases, depending on which criterion (ratio vs absolute number) is used for interpretation. However, increased gene dosage (>6 HER2 genes or HER2/CEP17 ratio >2.2), regardless of the evaluation method, is positively correlated with HER2 protein expression.

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

BACKGROUND: Chromosomal 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. METHODS: Virtual 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. RESULTS: Overall, 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. CONCLUSIONS: SNP 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.

Papillary renal cell-like carcinoma in a retroperitoneal teratoma.

We report a case of somatic type malignancy with papillary renal cell carcinoma differentiation arising in a retroperitoneal mixed germ cell tumor. The patient was a 36-year-old man with a synchronous mediastinal teratoma. The somatic type malignancy in the retroperitoneal tumor was composed of papillary structures covered by atypical epithelial cells with eosinophilic cytoplasm, prominent nucleoli and pseudostratified nuclei. Papillary cores contained numerous aggregates of foamy macrophages, typical of type I papillary renal cell carcinomas. The immunohistochemical profile was consistent with papillary renal cell carcinoma, including positive reactions for cytokeratin 7 and alpha-methyl acyl CoA racemase. There was no somatic type malignancy component in the synchronous mediastinal teratoma. Both the retroperitoneal and the mediastinal tumor showed gains of 12p and chromosome 17 material. There was no c-MET mutation in the somatic type malignancy. To our knowledge, this is the first report of a somatic type malignancy with features of papillary renal cell carcinoma arising in a germ cell tumor. It is important not to confuse such a retroperitoneal tumor with a conventional papillary renal cell carcinoma, because presence of other malignant histologies within the germ cell tumor may warrant different treatment. In such cases, the presence of isochromosome 12p can be helpful to the diagnosis.

Abnormal villous morphology associated with triple trisomy of paternal origin.

The vast majority of trisomies in spontaneous abortions (SAB) are single and of maternal origin, most frequently due to meiosis I errors. Triple trisomies are exceedingly rare (approximately 0.05% of spontaneous abortions), most often of maternal origin, and associated with increased maternal age. Some trisomic SAB specimens can exhibit abnormal villous morphology simulating a partial hydatidiform mole, a distinct form of hydatidiform mole characterized by diandric triploidy. A SAB specimen from a 27-year-old woman, G1P0 at 8 weeks gestational age, was reviewed in consultation to address the finding of morphological features suggestive of a partial hydatidiform mole but DNA ploidy analysis yielding a diploid result. The villi were irregularly shaped and hydropic but lacked trophoblastic hyperplasia; p57 expression was retained. Since fully developed features of a partial hydatidiform mole were lacking, additional analysis was performed. Molecular genotyping and single nucleotide polymorphism array analysis demonstrated biparental diploidy with trisomy of chromosomes 7, 13, and 20, all of paternal origin. The three trisomies may have originated from paternal meiosis II errors, or from mitotic nondisjunction. We believe this to be the first report of triple trisomy in a SAB confirmed to be of paternal origin.

EGFR and HER-2/neu expression in invasive apocrine carcinoma of the breast.

This study was undertaken to investigate epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2)/neu expression in a cohort of apocrine carcinomas of the breast with emphasis on the classification of the breast tumors with apocrine morphology. In total, 55 breast carcinomas morphologically diagnosed as apocrine were evaluated for the steroid receptor expression profile characteristic of normal apocrine epithelium (androgen receptor positive/estrogen receptor (ER) negative/progesterone receptor (PR) negative), and for the expression of EGFR and Her-2/neu proteins, and the copy number ratios of the genes EGFR/CEP7 and HER-2/CEP17. On the basis of the results of steroid receptors expression, 38 (69%) cases were classified as pure apocrine carcinoma (androgen receptor positive/ER negative/PR negative), whereas 17 (31%) were re-classified as apocrine-like carcinomas because they did not have the characteristic steroid receptor expression profile. Her-2/neu overexpression was observed in 54% of the cases (57% pure apocrine carcinomas vs 47% apocrine-like carcinomas). HER-2/neu gene amplification was demonstrated in 52% of all cases (54% pure apocrine carcinomas vs 46% apocrine-like carcinomas). EGFR protein (scores 1 to 3+) was detected in 62% of all cases and was expressed in a higher proportion of pure apocrine carcinomas than in the apocrine-like carcinomas group (76 vs 29%, P=0.006). In the pure apocrine carcinoma group, Her-2/neu and EGFR protein expression were inversely correlated (P=0.006, r=-0.499). EGFR gene amplification was observed in two pure apocrine carcinomas and one apocrine-like carcinoma. Polysomy 7 was commonly present in pure apocrine carcinomas (61 vs 27% of apocrine-like carcinomas; P=0.083) and showed a weak positive correlation with EGFR protein expression (P=0.025, r=0.326). Our study showed that apocrine breast carcinomas are molecularly diverse group of carcinomas. Strictly defined pure apocrine carcinomas are either HER-2-overexpressing breast carcinomas or triple-negative breast carcinomas, whereas apocrine-like carcinomas predominantly belong to the luminal phenotype. Pure apocrine carcinomas show consistent overexpression of either EGFR or HER-2/neu, which could have significant therapeutic implications.