Clinical Validation of a Circulating Tumor Cell Assay Using Density Centrifugation and Automated Immunofluorescence Microscopy.

OBJECTIVES: The US Food and Drug Administration (FDA)-approved CELLSEARCH assay (Menarini Silicon Biosystems) for circulating tumor cells (CTCs) relies on expression of an epithelial cell adhesion molecule to enrich for CTCs. We sought to validate a CTC assay (RareCyte) for clinical use that instead collects a buffy coat preparation enriched for CTCs. METHODS: Normal peripheral blood specimens spiked with cultured breast and prostate cancer cells and 47 clinical samples were used to validate assay performance. Specimens were enriched for buffy coat cells and applied onto 8 glass slides. The slides were immunofluorescently stained and imaged by automated microscopy and computer-aided image analysis. RESULTS: The assay was 100% specific for detecting spiked tumor cells. For samples spiked with 25, 50, and 125 cells, the percentage coefficients of variation were 42%, 21%, and 3.7%, respectively. Linearity studies demonstrated a slope of 0.99, an intercept of 1.6, and R2 of 0.96. Recoveries at the 25-, 50-, and 125-cell levels were 92%, 111%, and 100%, respectively. Clinical samples run on both CELLSEARCH and RareCyte correlated with an R2 of 0.8 after log-transformation and demonstrated 87.5% concordance using the CELLSEARCH criteria for predicting adverse outcomes. CONCLUSIONS: The RareCyte CTC assay has comparable performance to the FDA-cleared method and is ready for further clinical validation studies.

Evaluation of Exome Sequencing Criteria for Hospital Stewardship and Insurance Authorization at a Pediatric Hospital.

CONTEXT.­: Genomic molecular testing practices in a pediatric tertiary care institution can vary in utility by patient indication. OBJECTIVE.­: To evaluate exome sequencing (ES) ordering practices and the effects of applying criteria to support ES stewardship. Exome sequencing can provide molecular diagnostic information for patients with known or suspected genetic diseases, but it is relatively expensive, and the cost is often borne by patients, institutions, and payers. DESIGN.­: We examined ordering patterns of ES approved by board-certified geneticists at our tertiary pediatric care center, as well as preauthorization outcomes for ES requests. We compared positivity rates among patients by patient phenotype, composite insurance coverage criteria, and insurance preauthorization outcome. RESULTS.­: Patients who met composite coverage criteria were more likely to receive a positive result from ES compared to patients who did not meet composite coverage criteria, though this trend was not statistically significant. There was no significant difference in ES results between patients who were denied or not denied preauthorization by insurance payers. CONCLUSIONS.­: Insurance payers should consider implementing and/or expanding coverage criteria for ES, and institutions should implement stewardship programs to support appropriate ES practices.

Validating cell-free DNA from supernatant for molecular diagnostics on cytology specimens.

BACKGROUND: Cytology specimens are often used for biomarker testing in the setting of neoplasia. On occasion, formalin-fixed paraffin-embedded (FFPE) cell blocks unfortunately may not yield sufficient material for testing. Recent studies have suggested that residual supernatant fluid from cell block preparation is a valuable source of DNA: both cellular and cell-free DNA (cfDNA). In the present study, the use of cfDNA from supernatant is compared against DNA from FFPE materials. METHODS: cfDNA was extracted prospectively from residual supernatants of 30 cytology samples (29 neoplastic cases and 1 benign ascitic fluid from a patient with a history of melanoma). Samples were tested using clinically validated next-generation-sequencing platforms and the results were compared with data from paired FFPE cell blocks in a real-time prospective clinical setting. Thirteen samples were tested on an amplicon-based assay (Solid Tumor Hotspot), and 17 samples were tested using a comprehensive capture-based assay (UW-Oncoplex). RESULTS: Neoplastic content was estimated by mutational variant allele fraction, with a mean content of 24.0% and 25.8% in supernatant and FFPE, respectively. The variant concordance between paired samples was 90%, and identical results were detected in both supernatant and FFPE samples in 74% of cases. CONCLUSIONS: This study confirmed that cfDNA from supernatant is a viable alternative to FFPE cell blocks for molecular biomarker testing using both amplicon-based and capture-based assays with potential for decreasing additional tissue sampling and faster turnaround time.

An Alternate Diagnostic Algorithm for the Diagnosis of Intraepithelial Fallopian Tube Lesions.

Intraepithelial fallopian tube neoplasia is thought to be a precursor lesion to high-grade serous carcinoma of the Müllerian adnexae, particularly in women with BRCA1 or BRCA2 mutations. This association has led to recommendations to assess fallopian tubes for intraepithelial atypia. However, the diagnostic reproducibility of a diagnosis of intraepithelial neoplasia is unclear. In this study, 2 gynecologic pathologists independently evaluated sections of fallopian tubes from a sample of women (N=198, 623 slides) undergoing salpingectomy. A total of 101 (54%) women were undergoing risk-reducing salpingo-oophorectomy. Pathologists were blinded to patient histories and prior diagnoses. Pathologists rendered one of three diagnoses for each slide: "negative for fallopian tube intraepithelial neoplasia (FTIN)," "indeterminate for FTIN," or "definite for FTIN." Cases that were considered by histology definite for FTIN or suspicious for FTIN were stained with p53 and Ki67. Pathologists agreed on the diagnosis of "definite for FTIN" 61.5% of the time. There was no agreement on any cases for the diagnosis of "indeterminate for FTIN." Fifteen "indeterminate for FTIN" and 12 "definite for FTIN" cases were stained with p53 and Ki67. Two of the "indeterminate" cases (13%) had p53-positive foci. Five of the "definite" cases had p53-positive foci. In 3 of the other 8 "definite" cases, there was obvious carcinoma present, but the carcinoma did not stain with p53, suggesting a possible null phenotype. We propose that immunostains should only be used to aid in the diagnosis of FTIN in cases with indeterminate histology. The use of p53 immunohistochemistry in cases that were considered "definite for FTIN" by histology was minimally helpful, and in fact often served to further confuse the diagnosis.

Should Margin Status of Biopsies Be Included in Dermatopathology Reports? Clinician Preferences and the Importance of Unambiguous Language.

Significant communication occurs between pathologists and clinicians through the dermatopathology report. Our objective was to describe clinician preference about reporting of the margin status of skin biopsies of nonmelanoma skin cancers. An anonymous survey was sent to 243 medical providers who submitted specimens to a single institution university medical center; 50 complete responses from attending-level providers and advance practice providers were received. The majority (96%) of those surveyed indicated margins should be reported on skin biopsies of neoplasms, particularly nonmelanoma skin cancers (basal cell carcinoma 96% and squamous cell carcinoma 92%) and atypical nevi (96%). When asked about particular language used to describe the margin status, some phrasing led to more variance in respondents' clinical management decisions, with 96%-98% of respondents making the same decision when presented with "unambiguous" terms and 58%-84% of respondents making the same decision when presented with "ambiguous" language (P < 0.001). Respondents generally preferred "unambiguous" margin descriptions when shown an involved margin (70% vs. 30%, P < 0.001) but accepted "ambiguous" language when the margin was clearly uninvolved (68% vs. 32%, P = 0.015). Most respondents (88%) desire inclusion of treatment recommendations in dermatopathology reports. Microscopic descriptions were highly utilized, particularly by nondermatology trained clinicians (97% vs. 80%, P = 0.09). Clinicians desire inclusion of margins for skin biopsies in dermatopathology reports, at least in some circumstances. The choice of language used to describe the margin status in dermatopathology reports has important implications for patient care. Margin descriptors that are unclear or ambiguous may lead to more variance in clinical management.

Medical renal diseases are frequent but often unrecognized in adult autopsies.

Kidney diseases affect many hospitalized patients and contribute to morbidity and mortality. Therefore, kidney disease should be prevalent, but the frequency and spectrum of medical renal pathology in autopsy specimens has not been well documented. We sought to determine the spectrum of medical renal pathology in adult autopsy specimens and the frequency of overlooked diagnoses. We reviewed the hematoxylin- and eosin-stained kidney sections from 140 adult autopsies performed at a large teaching hospital over a 2-year period. Fifty-eight cases (41%) had findings warranting further analysis, including alterations in glomerular matrix and/or cellularity, atypical or pigmented casts, thrombi, tubulointerstitial or vascular inflammation, or deposition of amorphous material. After additional studies and clinical correlation, the pathologic changes in 43 cases (31%) were categorized as follows: diabetic nephropathy, bile cast nephropathy, thrombotic microangiopathy, infection-related glomerulonephritis, focal necrotizing/crescentic glomerulonephritis, oxalate nephropathy, light-chain cast nephropathy, amyloidosis, urate nephropathy, hemosiderosis, antineutrophil cytoplasmic antibody-associated vasculitis, polyoma virus nephropathy, atheroembolic disease, and nephrocalcinosis. These diagnoses were not reported in 26 (60%) cases during the initial autopsy evaluation. This study demonstrates that medical renal diseases are common in autopsy cases, but significant diagnoses can be easily overlooked. Autopsy kidney specimens are a rich source of renal pathology and their evaluation should be emphasized in anatomic pathology residency training. Ultimately, our understanding of how kidney disease contributes to morbidity and mortality will benefit from accurate recognition of renal pathology in autopsy specimens.

An online monogenic diabetes discussion group: supporting families and fueling new research.

Many online support groups are available for patients with rare disorders, but scant evidence is available on how effectively such groups provide useful information or valuable psychosocial support to their participants. It is also unclear to what extent physicians and researchers may learn more about these disorders by participating in such groups. To formally assess the utility of the Kovler Monogenic Diabetes Registry online discussion group for patients and families affected by KATP channel-related monogenic neonatal diabetes in providing psychosocial and informational support and in identifying concerns unique to patients with this rare form of diabetes. We qualitatively analyzed all 1,410 messages from the online group that consisted of 64 participants affected by KATP channel monogenic diabetes and 11 researchers. We utilized the Social Behavior Support Code to assign each message to a support category and deductive thematic analysis to identify discussion topics addressed by each message. 44% of messages provided/requested informational support, whereas 31.4% of the messages contained psychosocial/emotional support. The most popular topics of postings to the forums were diabetes treatment (503 messages) and neurodevelopmental concerns (472 messages). Participation in the discussion led researchers to modify survey instruments and design new studies focusing on specific topics of concern, such as sleep. We demonstrate that an online support group for a monogenic form of diabetes is an effective informational tool that also provides psychosocial support. Participation by researchers and care providers can inform future research directions and highlight issues of patient concern.

Continued lessons from the INS gene: an intronic mutation causing diabetes through a novel mechanism.

BACKGROUND: Diabetes in neonates usually has a monogenic aetiology; however, the cause remains unknown in 20-30%. Heterozygous INS mutations represent one of the most common gene causes of neonatal diabetes mellitus. METHODS: Clinical and functional characterisation of a novel homozygous intronic mutation (c.187+241G>A) in the insulin gene in a child identified through the Monogenic Diabetes Registry (http://monogenicdiabetes.uchicago.edu). RESULTS: The proband had insulin-requiring diabetes from birth. Ultrasonography revealed a structurally normal pancreas and C-peptide was undetectable despite readily detectable amylin, suggesting the presence of dysfunctional ß cells. Whole-exome sequencing revealed the novel mutation. In silico analysis predicted a mutant mRNA product resulting from preferential recognition of a newly created splice site. Wild-type and mutant human insulin gene constructs were derived and transiently expressed in INS-1 cells. We confirmed the predicted transcript and found an additional transcript created via an ectopic splice acceptor site. CONCLUSIONS: Dominant INS mutations cause diabetes via a mutated translational product causing endoplasmic reticulum stress. We describe a novel mechanism of diabetes, without ß cell death, due to creation of two unstable mutant transcripts predicted to undergo nonsense and non-stop-mediated decay, respectively. Our discovery may have broader implications for those with insulin deficiency later in life.