Lawrence J. Jennings

Recommended principles and practices for validating clinical molecular pathology tests.

CONTEXT The use of DNA- and RNA-based tests continues to grow for applications as varied as inherited disease, infectious disease, cancer, identity testing, human leukocyte antigen typing, and pharmacogenetics. Progress is driven in part by the huge growth in knowledge about the molecular basis of disease coupled with advancements in technologic capabilities. In addition to requirements for clinical utility, every molecular test also may have limitations that must be carefully considered before clinical implementation. Analytic and clinical performance characteristics as well as test limitations are established and documented through the process of test validation. OBJECTIVE To describe the established principles of test validation, along with relevant regulations in the United States, in order to provide a rational approach to introducing molecular tests into the clinical laboratory. DATA SOURCES PubMed review of published literature, published guidelines, and online information from national and international professional organizations. CONCLUSIONS These resources and recommendations provide a framework for validating clinical tests.

Congenital central hypoventilation syndrome from past to future: Model for translational and transitional autonomic medicine

The modern story of CCHS began in 1970 with the first description by Mellins et al., came most visibly to the public eye with the ATS Statement in 1999, and continues with increasingly fast paced advances in genetics. Affected individuals have diffuse autonomic nervous system dysregulation (ANSD). The paired‐like homeobox gene PHOX2B is the disease‐defining gene for CCHS; a mutation in the PHOX2B gene is requisite to the diagnosis of CCHS. Approximately 90% of individuals with the CCHS phenotype will be heterozygous for a polyalanine repeat expansion mutation (PARM); the normal allele will have 20 alanines and the affected allele will have 24–33 alanines (genotypes 20/24–20/33). The remaining ∼10% of individuals with CCHS will have a non‐PARM (NPARM), in the PHOX2B gene; these will be missense, nonsense, or frameshift. CCHS and PHOX2B are inherited in an autosomal dominant manner with a stable mutation. Approximately 8% of parents of a CCHS proband will be mosaic for the PHOX2B mutation. A growing number of cases of CCHS are identified after the newborn period, with presentation from infancy into adulthood. An improved understanding of the molecular basis of the PHOX2B mutations and of the PHOX2B genotype/CCHS phenotype relationship will allow physicians to anticipate the clinical phenotype for each affected individual. To best convey the remarkable history of CCHS, and to describe the value of recognizing CCHS as a model for translational and transitional autonomic medicine, we present this review article in the format of a chronological story, from 1970 to the present day. Pediatr Pulmonol. 2009; 44:521–535.

Detection and Quantification of BCR-ABL1 Fusion Transcripts by Droplet Digital PCR

Monitoring BCR-ABL1 fusion transcripts by real-time quantitative RT-PCR has become an important clinical test for the management of patients with chronic myeloid leukemia. However, it has some inherent limitations with regard to its lower limit of detection and limit of quantification. Improvement in the lower limit of detection could aid clinicians in selecting candidates for discontinuation of tyrosine kinase inhibitors without relapse. Improvement in the limit of quantification may also avoid unnecessary testing or changes in therapy. Here, we demonstrate the advantages of droplet digital RT-PCR with regard to simplicity, lower limit of detection, and limit of quantification. We expect the advantages of droplet digital RT-PCR will make it the preferred method for quantification of BCR-ABL1 fusion transcripts.

Guidelines for Validation of Next-Generation Sequencing–Based Oncology Panels: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists

Next-generation sequencing (NGS) methods for cancer testing have been rapidly adopted by clinical laboratories. To establish analytical validation best practice guidelines for NGS gene panel testing of somatic variants, a working group was convened by the Association of Molecular Pathology with liaison representation from the College of American Pathologists. These joint consensus recommendations address NGS test development, optimization, and validation, including recommendations on panel content selection and rationale for optimization and familiarization phase conducted before test validation; utilization of reference cell lines and reference materials for evaluation of assay performance; determining of positive percentage agreement and positive predictive value for each variant type; and requirements for minimal depth of coverage and minimum number of samples that should be used to establish test performance characteristics. The recommendations emphasize the role of laboratory director in using an error-based approach that identifies potential sources of errors that may occur throughout the analytical process and addressing these potential errors through test design, method validation, or quality controls so that no harm comes to the patient. The recommendations contained herein are intended to assist clinical laboratories with the validation and ongoing monitoring of NGS testing for detection of somatic variants and to ensure high quality of sequencing results.

Gain of 1q is associated with inferior event-free and overall survival in patients with favorable histology Wilms tumor: a report from the Children's Oncology Group.

Wilms tumor is the most common childhood renal tumor. Although the majority of patients with favorable histology Wilms tumor (FHWT) have good outcomes, some patients still experience disease recurrence and death from disease. The goal of the current study was to determine whether tumor‐specific chromosome 1q gain is associated with event‐free survival (EFS) and overall survival (OS) in patients with FHWT.

A prospective, multi-Institutional diagnostic trial to determine pathologist accuracy in estimation of percentage of malignant cells

CONTEXT The fraction of malignant cells in tumor tissue submitted for tests of genetic alterations is a critical variable in testing accuracy. That fraction is currently determined by pathologist visual estimation of the percentage of malignant cells. Inaccuracy could lead to a false-negative test result. OBJECTIVE To describe a prospective, multi-institutional study to determine pathologist estimation accuracy. DESIGN Ten ×20 magnification images of hematoxylin-eosin-stained colon tissue specimens were sent as an educational component of the College of American Pathologists KRAS-B 2011 Survey. Data from 194 labs were analyzed and compared to a criterion standard with comprehensive manual nuclear counts. RESULTS Survey responses indicated low interlaboratory precision of pathologist estimation, but mean estimates were fairly accurate. A total of 5 of the 10 cases assessed showed more than 10% of respondents overestimating in a manner that could lead to false-negative test results. CONCLUSIONS The significance of estimation errors resulting in molecular testing failures with implications for patient care is unknown, but the current study suggests false-negative test results may occur.

Mammary analogue secretory carcinoma of the parotid gland in a pediatric patient

S alivary neoplasms comprise 1% to 3% of all head and neck malignancies. Less than 5% of salivary gland malignancies are diagnosed in children. A recent study in adult patients describes a new salivary gland tumor, mammary analogue secretory carcinoma of the salivary gland (MASC). This lesion has pathological characteristics similar to secretory carcinoma of the breast, salivary acinic cell carcinoma, and low-grade cystadenocarcinoma. MASC is frequently associated with a translocation, t(12;15)(p13;q25), resulting in the fusion gene ETV6-NTRK3. This translocation has been demonstrated consistently in secretory carcinoma of the breast. The tyrosine kinase encoded by this fusion gene has been directly related to transformation of epithelial cells in mouse mammary glands. Children’s Memorial Hospital institutional review board approval was obtained, and we report the first case of MASC in the parotid gland of a child.

WT1 Mutation and 11P15 Loss of Heterozygosity Predict Relapse in Very Low-Risk Wilms Tumors Treated With Surgery Alone: A Children's Oncology Group Study

PURPOSE Childrens Oncology Group defines very low-risk Wilms tumors (VLRWT) as stage I favorable histology Wilms tumors weighing less than 550 g in children younger than 24 months of age. VLRWTs may be treated with nephrectomy alone. However, 10% to 15% of VLRWTs relapse without chemotherapy. Previous studies suggest that VLRWTs with low WT1 expression and/or 11p15 loss of heterozygosity (LOH) may have increased risk of relapse. The current study validates these findings within prospectively identified children with VLRWT who did not receive adjuvant chemotherapy. PATIENTS AND METHODS Fifty-six VLRWTs (10 relapses) were analyzed for mutation of WT1, CTNNB1, and WTX; for 11p15 LOH using microsatellite analysis; and for H19DMR and KvDMR1 methylation. RESULTS 11p15 LOH was identified in 19 (41%) of 46 evaluable VLRWTs and was significantly associated with relapse (P < .001); 16 of 19 were isodisomic for 11p15. WT1 mutation was identified in nine (20%) of 45 evaluable VLRWTs and was significantly associated with relapse (P = .004); all nine cases also had 11p15 LOH. All evaluable tumors showing LOH by microsatellite analysis also showed LOH by methylation analysis. Retention of the normal imprinting pattern was identified in 24 of 42 evaluable tumors, and none relapsed. Loss of imprinting at 11p15 was identified in one of 42 tumors. CONCLUSION WT1 mutation and 11p15 LOH are associated with relapse in patients with VLRWTs who do not receive chemotherapy. These may provide meaningful biomarkers to stratify patients for reduced chemotherapy in the future. VLRWTs show a different incidence of WT1 mutation and 11p15 imprinting patterns than has been reported in Wilms tumors of all ages.

Use of Porcine Acellular Dermal Matrix as a Dermal Substitute in Rats

ObjectiveTo examine porcine acellular dermal matrix (ADM) as a xenogenic dermal substitute in a rat model. Summary Background DataAcellular dermal matrix has been used in the treatment of full-thickness skin injuries as an allogenic dermal substitute providing a stable wound base in human and animal studies. MethodsXenogenic and allogenic ADMs were produced by treating porcine or rat skin with Dispase and Triton X-100. Full-thickness skin defects (225 mm2) were created on the dorsum of rats (n = 29), porcine or rat ADMs were implanted in them, and these were overlain with ultrathin split-thickness skin grafts (STSGs). In two adjacent wounds, 0.005- or 0.017-inch-thick autografts were implanted. In other experiments, the antimicrobial agent used during ADM processing (azide or a mixture of antibiotics) and the orientation of the implanted ADM (papillary or reticular side of ADM facing the STSG) were studied. Grafts were evaluated grossly and histologically for 30 days after surgery. ResultsSignificant wound contraction was seen at 14, 20, and 30 days after surgery in wounds receiving xenogenic ADM, allogenic ADM, and thin STSGs. Contraction of wounds containing xenogenic ADM was significantly greater than that of wounds containing allogenic ADM at 30 days after surgery. Graft take was poor in wounds containing xenogenic ADM and moderately good in those containing allogenic ADM. Wound healing was not significantly affected by the antimicrobial agent used during ADM preparation or by the ADM orientation. ConclusionDispase–Triton-treated allogenic ADM was useful as a dermal substitute in full-thickness skin defects, but healing with xenogenic ADM was poor.

Variable human phenotype associated with novel deletions of the PHOX2B gene.

Clinical testing for PHOX2B mutations is widely used for patients with any symptoms suggestive of hypoventilation (with/without anatomic/physiologic autonomic dysregulation), though not necessarily with the congenital central hypoventilation syndrome (CCHS) phenotype. Consequently, a multitude of referrals for clinical PHOX2B testing (fragment analysis of the 20 polyalanine repeat region and/or sequencing of entire coding region) have no identifiable mutation. Whole gene deletions/duplications have recently been identified as a common disease‐causing mechanism, but have not been reported in a clinical population referred for PHOX2B testing. The objective of this study was to determine if PHOX2B exon or whole gene deletion/duplication would be identified in a subset of patients referred for PHOX2B testing.