Deep sequencing with intronic capture enables identification of an APC exon 10 inversion in a patient with polyposis.

PURPOSE: Single-exon inversions have rarely been described in clinical syndromes and are challenging to detect using Sanger sequencing. We report the case of a 40-year-old woman with adenomatous colon polyps too numerous to count and who had a complex inversion spanning the entire exon 10 in APC (the gene encoding for adenomatous polyposis coli), causing exon skipping and resulting in a frameshift and premature protein truncation. METHODS: In this study, we employed complete APC gene sequencing using high-coverage next-generation sequencing by ColoSeq, analysis with BreakDancer and SLOPE software, and confirmatory transcript analysis. RESULTS: ColoSeq identified a complex small genomic rearrangement consisting of an inversion that results in translational skipping of exon 10 in the APC gene. This mutation would not have been detected by traditional sequencing or gene-dosage methods. CONCLUSION: We report a case of adenomatous polyposis resulting from a complex single-exon inversion. Our report highlights the benefits of large-scale sequencing methods that capture intronic sequences with high enough depth of coverage-as well as the use of informatics tools-to enable detection of small pathogenic structural rearrangements.

Clinical utility of a circulating tumor cell assay in Merkel cell carcinoma.

BACKGROUND: Quantitation of circulating tumor cells (CTCs) has utility in managing breast, colon, and prostate carcinomas. OBJECTIVE: We sought to determine whether a commercially available CTC assay provides prognostic information in Merkel cell carcinoma (MCC), insight into treatment responses, or both. METHODS: We analyzed CTCs in 52 specimens from 34 patients with MCC. RESULTS: The presence of CTCs correlated with extent of disease at blood draw (P = .004). Among 15 patients with regional nodal disease, CTC-negative patients had 80% disease-specific survival at 2 years after the test, versus 29% for CTC-positive patients (P = .015). Among the entire cohort, those without CTCs had 72% MCC-specific survival whereas CTC-positive patients had 25% survival (n = 34, median follow-up 19 months, P = .0003). Fifty seven percent of patients with MCC had a cytokeratin "dot" visible in 20% or more of CTCs, a feature that was absent among CTCs from other carcinomas (0 of 13 cases). LIMITATIONS: CTC assay was performed at variable times after diagnosis and heterogeneity in extent of disease affects interpretability of the data. CONCLUSION: CTC detection in MCC is feasible and appears to add prognostic information, particularly in patients with regional nodal disease. It may also assist clinical management in certain situations, including differentiating metastatic MCC cells from those of other carcinomas.

Validation and implementation of targeted capture and sequencing for the detection of actionable mutation, copy number variation, and gene rearrangement in clinical cancer specimens.

Recent years have seen development and implementation of anticancer therapies targeted to particular gene mutations, but methods to assay clinical cancer specimens in a comprehensive way for the critical mutations remain underdeveloped. We have developed UW-OncoPlex, a clinical molecular diagnostic assay to provide simultaneous deep-sequencing information, based on >500× average coverage, for all classes of mutations in 194 clinically relevant genes. To validate UW-OncoPlex, we tested 98 previously characterized clinical tumor specimens from 10 different cancer types, including 41 formalin-fixed paraffin-embedded tissue samples. Mixing studies indicated reliable mutation detection in samples with ≥ 10% tumor cells. In clinical samples with ≥ 10% tumor cells, UW-OncoPlex correctly identified 129 of 130 known mutations [sensitivity 99.2%, (95% CI, 95.8%-99.9%)], including single nucleotide variants, small insertions and deletions, internal tandem duplications, gene copy number gains and amplifications, gene copy losses, chromosomal gains and losses, and actionable genomic rearrangements, including ALK-EML4, ROS1, PML-RARA, and BCR-ABL. In the same samples, the assay also identified actionable point mutations in genes not previously analyzed and novel gene rearrangements of MLL and GRIK4 in melanoma, and of ASXL1, PIK3R1, and SGCZ in acute myeloid leukemia. To best guide existing and emerging treatment regimens and facilitate integration of genomic testing with patient care, we developed a framework for data analysis, decision support, and reporting clinically actionable results.

A mosaic PTEN mutation causing Cowden syndrome identified by deep sequencing.

PURPOSE: Mosaic PTEN mutations are not well described in Cowden syndrome. We report a 40-year-old woman with a clinical diagnosis of Cowden syndrome including Lhermitte-Duclos disease, who had a mosaic PTEN mutation detected by next-generation deep sequencing. METHODS: Complete PTEN gene sequencing by the Sanger method and deletion/duplication analysis performed on DNA extracted from blood leukocytes at a commercial clinical laboratory did not identify a mutation. Because of high suspicion of a PTEN mutation, we repeated testing by next-generation sequencing using the ColoSeq assay, which sequences the entire PTEN locus at >320-fold average coverage. RESULTS: ColoSeq identified a frameshift PTEN mutation (c.767_768delAG) in 1.7% of sequencing reads from peripheral blood leukocytes (21/1,184 reads), which is below the limit of detection of most Sanger sequencing methods. The mutation was detected at full heterozygous levels in skin fibroblasts and a cerebellar tumor, and at approximately the 25% level in colonic and endocervical mucosa, confirming somatic mosaicism. CONCLUSION: Our report highlights the power of deep next-generation sequencing to identify mosaic mutations that can be missed by traditional less sensitive approaches. We speculate that mosaic PTEN mutations are more common in Cowden syndrome than previously described.

An automated high-throughput counting method for screening circulating tumor cells in peripheral blood.

Enumeration of circulating tumor cells (CTCs) has proved valuable for early detection and prognosis in cancer treatment. This paper describes an automated high-throughput counting method for CTCs based on microfluidics and line-confocal microscopy. Peripheral blood was directly labeled with multiple antibodies, each conjugated with a different fluorophore, pneumatically pumped through a microfluidic channel, and interrogated by a line-confocal microscope. On the basis of the fluorescence signals and labeling schemes, the count of CTCs was automatically reported. Due to the high flow rate, 1 mL of whole blood can be analyzed in less than 30 min. We applied this method in analyzing CTCs from 90 stage IV breast cancer patient samples and performed a side-by-side comparison with the results of the CellSearch assay, which is the only method approved by the U.S. Food and Drug Administration at present for enumeration of CTCs. This method has a recovery rate for cultured breast cancer cells of 94% (n = 9), with an average of 1.2 counts/mL of background level of detected CTCs from healthy donors. It detected CTCs from breast cancer patients ranging from 15 to 3375 counts/7.5 mL. Using this method, we also demonstrate the ability to enumerate CTCs from breast cancer patients that were positive for Her2 or CD44(+)/CD24(-), which is a putative cancer stem cell marker. This automated method can enumerate CTCs from peripheral blood with high throughput and sensitivity. It could potentially benefit the clinical diagnosis and prognosis of cancer.

Primary failure of eruption: further characterization of a rare eruption disorder.

INTRODUCTION: Posterior open bite has several possible causes, including primary failure of eruption (PFE) that affects all teeth distal to the most mesial involved tooth, mechanical failure of eruption (MFE) (primarily ankylosis) that affects only the involved tooth or teeth, and soft-tissue interferences with eruption (other). METHODS: Radiographs and other clinical records for 97 cases of failure of posterior eruption submitted for consultation were analyzed to further characterize PFE and distinguish it from MFE. RESULTS: Of the 97 cases, 38 were judged to be clear-cut PFE; 19 were diagnosed as MFE; 32 were classified as indeterminate failure because they were too young to be certain of the distinction between PFE and MFE; and 8 were placed in the "other" category. Two subtypes of PFE were observed. In type 1, eruption failure occurred at or near the same time for all teeth in an affected quadrant. In type 2, a gradient of the time of failure was present, so that some further development of the teeth posterior to the most mesial affected tooth was observed before eruption failure. A family history of eruption problems was noted in 10 of the 38 PFE subjects (26%), and a pedigree analysis was done for 4 families. This was consistent with autosomal dominant transmission. CONCLUSIONS: The distinction between PFE and MFE is clinically important because it determines whether all posterior teeth, or only individual affected teeth, will not respond to orthodontic force. Certain diagnosis often requires progress radiographs so that the pattern of eruption of teeth distal to the most mesial affected tooth can be observed.