Daynna J. Wolff

Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists

Widespread clinical laboratory implementation of next-generation sequencing-based cancer testing has highlighted the importance and potential benefits of standardizing the interpretation and reporting of molecular results among laboratories. A multidisciplinary working group tasked to assess the current status of next-generation sequencing-based cancer testing and establish standardized consensus classification, annotation, interpretation, and reporting conventions for somatic sequence variants was convened by the Association for Molecular Pathology with liaison representation from the American College of Medical Genetics and Genomics, American Society of Clinical Oncology, and College of American Pathologists. On the basis of the results of professional surveys, literature review, and the Working Groups subject matter expert consensus, a four-tiered system to categorize somatic sequence variations based on their clinical significances is proposed: tier I, variants with strong clinical significance; tier II, variants with potential clinical significance; tier III, variants of unknown clinical significance; and tier IV, variants deemed benign or likely benign. Cancer genomics is a rapidly evolving field; therefore, the clinical significance of any variant in therapy, diagnosis, or prognosis should be reevaluated on an ongoing basis. Reporting of genomic variants should follow standard nomenclature, with testing method and limitations clearly described. Clinical recommendations should be concise and correlate with histological and clinical findings.

Fluorescence in situ hybridization for detecting transitional cell carcinoma: implications for clinical practice.

To determine the diagnostic sensitivity of genetic studies using fluorescence in situ hybridization (FISH) for detecting both new and recurrent cases of transitional cell carcinoma (TCC) in a routine clinical practice setting, as bladder cancer has a significant risk of recurrence and progression to invasive disease and thus sensitive surveillance testing is very important.

American College of Medical Genetics recommendations for the design and performance expectations for clinical genomic copy number microarrays intended for use in the postnatal setting for detection of constitutional abnormalities

Genomic copy number microarrays have significantly increased the diagnostic yield over a karyotype for clinically significant imbalances in individuals with developmental delay, intellectual disability, multiple congenital anomalies, and autism, and they are now accepted as a first tier diagnostic test for these indications. As it is not feasible to validate microarray technology that targets the entire genome in the same manner as an assay that targets a specific gene or syndromic region, a new paradigm of validation and regulation is needed to regulate this important diagnostic technology. We suggest that these microarray platforms be evaluated and manufacturers regulated for the ability to accurately measure copy number gains or losses in DNA (analytical validation) and that the subsequent interpretation of the findings and assignment of clinical significance be determined by medical professionals with appropriate training and certification. To this end, the American College of Medical Genetics, as the professional organization of board-certified clinical laboratory geneticists, herein outlines recommendations for the design and performance expectations for clinical genomic copy number microarrays and associated software intended for use in the postnatal setting for detection of constitutional abnormalities.

Laboratory guideline for Turner syndrome

Turner syndrome is a disorder that has distinct clinical features and has karyotypic aberrations with loss of critical regions of the X chromosome. Several clinical guidelines on the diagnosis and management of patients with Turner syndrome have been published, but there is relatively little on the laboratory aspects associated with this disorder. This disease-specific laboratory guideline provides laboratory guidance for the diagnosis/study of patients with Turner syndrome and its variants. Because the diagnosis of Turner syndrome involves both a clinical and laboratory component, both sets of guidelines are required for the provision of optimal care for patients with Turner syndrome.

Evaluation of urovysion and cytology for bladder cancer detection: a study of 1835 paired urine samples with clinical and histologic correlation.

Urine cytology has been used for screening of bladder cancer but has been limited by its low sensitivity. UroVysion is a multiprobe fluorescence in situ hybridization (FISH) assay that detects common chromosome abnormalities in bladder cancers. For this study, the authors evaluated the effectiveness of multiprobe FISH and urine cytology in detecting urothelial cell carcinoma (UCC) in the same urine sample.

Deletions in Xq26.3–q27.3 including FMR1 result in a severe phenotype in a male and variable phenotypes in females depending upon the X inactivation pattern

Abstract High resolution cytogenetics, microsatellite marker analyses, and fluorescence in situ hybridization were used to define Xq deletions encompassing the fragile X gene, FMR1, detected in individuals from two unrelated families. In Family 1, a 19-year-old male had facial features consistent with fragile X syndrome; however, his profound mental and growth retardation, small testes, and lover limb skeletal defects and contractures demonstrated a more severe phenotype, suggestive of a contiguous gene syndrome. A cytogenetic deletion including Xq26.3–q27.3 was observed in the proband, his phenotypically normal mother, and his learning-disabled non-dysmorphic sister. Methylation analyses at the FMR1 and androgen receptor loci indicated that the deleted X was inactive in > 95% of his mother’s white blood cells and 80–85% of the sister’s leukocytes. The proximal breakpoint for the deletion was approximately 10 Mb centromeric to FMR1, and the distal breakpoint mapped 1 Mb distal to FMR1. This deletion, encompassing ∼13 Mb of DNA, is the largest deletion including FMR1 reported to date. In the second family, a slightly smaller deletion was detected. A female with moderate to severe mental retardation, seizures, and hypothyroidism, had a de novo cytogenetic deletion extending from Xq26.3 to q27.3, which removed ∼12 Mb of DNA around the FMR1 gene. Cytogenetic and molecular data revealed that ∼50% of her white blood cells contained an active deleted X. These findings indicate that males with deletions including Xq26.3–q27.3 may exhibit a more severe phenotype than typical fragile X males, and females with similar deletions may have an abnormal phenotype if the deleted X remains active in a significant proportion of the cells. Thus, important genes for intellectual and neurological development, in addition to FMR1, may reside in Xq26.3–q27.3. One candidate gene in this region, SOX3, is thought to be involved in neuronal development and its loss may partly explain the more severe phenotypes of our patients.

Multi-Site PCR-based CMV viral load assessment-assays demonstrate linearity and precision, but lack numeric standardization: a report of the association for molecular pathology.

Viralload (VL) assessment of cytomegalovirus (CMV) by real-time PCR is an important tool for diagnosing and monitoring CMV viremia in patients with compromised immune systems. We report results from a sample exchange organized by members of the Association for Molecular Pathology that compared PCR results from 23 laboratories; 22 such laboratories used a laboratory-developed real-time PCR assay and one laboratory used a competitive PCR assay. The samples sent to each laboratory were comprised of a dilution panel of CMV virion-derived reference materials that ranged from 0 to 500,000 copies/ml. Accuracy, linearity, and intralaboratory precision were established for the different laboratory-developed assays. Overall, PCR results were linear for each laboratory (R(2) > 0.97 in all but two). While 13 laboratories showed no significant quantitative assay bias, 10 laboratories reported VLs that were significantly different compared with expected values (bias range, -0.82 to 1.4 logs). The intralaboratory precision [mean coefficient of variance of 2% to 5% (log-scale)] suggested that changes in VLs of less than 3- to fivefold may not be significantly different. There was no significant association between laboratory-specific technical variables (PCR platform, calibrator, extraction method) and assay linearity or accuracy. These data suggested that, within each laboratory, relative VL values were linear, but additional method standardization and a CMV DNA reference standard are needed to allow laboratories to achieve comparable numeric results.

Redefining the risks of prenatally ascertained supernumerary marker chromosomes: a collaborative study

Background: A marker chromosome is defined as a structurally abnormal chromosome that cannot be identified by routine cytogenetics. The risk for phenotypic abnormalities associated with a marker chromosome depends on several factors, including inheritance, mode of ascertainment, chromosomal origin, and the morphology, content, and structure of the marker. Methods: to understand the karyotype-phenotype relationship of prenatally ascertained supernumerary de novo marker chromosomes, we combined data from prenatal cases obtained from 12 laboratories with those from studies in the literature. We were able to obtain cytogenetic and phenotypic data from 108 prenatally ascertained supernumerary de novo marker chromosomes to refine the phenotypic risk associated with these markers. Because of the growing number of cases and because more techniques are available to delineate marker morphology, we have been able to group risk estimates into subcategories, such as by marker type and whether there are ultrasound abnormalities. Results: If a de novo supernumerary marker chromosome is found prenatally, our data suggest there is a 26% risk for phenotypic abnormality when there is no other information defining the marker (such as chromosomal origin or information about the existing phenotype). However, if high resolution ultrasound studies are normal, this risk reduces to 18%. Conclusions: Our findings strongly support the value of additional genetic studies for more precisely defining the risk in individual cases involving marker chromosomes.

Developing a sustainable process to provide quality control materials for genetic testing

Purpose: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community.Methods: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps.Results: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step.Conclusions: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

Pilot assessment of the subtelomeric regions of children with autism: Detection of a 2q deletion

Purpose: Autism is a chronic neurodevelopmental disorder characterized by deficits in reciprocal social interaction, language and communication, and by the presence of stereotypical behaviors. The disorder is a complex genetic trait with no known predisposing genes. We report the results of a pilot project to screen for aberrations in the gene-rich subtelomeric chromosomal regions of a cohort of children with autism.Methods: For our pilot project, we used a multiprobe system that includes probes for the subtelomeric regions of all human chromosomes. We assessed the subtelomeric regions of chromosomes from 10 children with a diagnosis of autism.Results: The screen identified one child with an apparent deletion of the subtelomeric region of chromosome 2q; nine children and pooled control samples yielded normal results. The deletion in our patient was confirmed with two other subtelomeric probes and a targeted cytogenetic study revealed a subtle difference in appearance for one chromosome 2 homologue.Conclusion: There have been several reports of children with dysmorphic features, autistic behaviors, and 2q deletions detectable with standard cytogenetic techniques. It may be that the distal region of chromosome 2q harbors a gene or genes that may predispose to autism.