Karen M. Koehler

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.

Sensitive and High-Throughput Isolation of Rare Cells from Peripheral Blood with Ensemble-Decision Aliquot Ranking†

This paper describes an approach called ensemble decision aliquot ranking (eDAR) for isolating rare cells from peripheral blood. eDAR has a recovery of over 93% (n=9) with a zero false positive rate (n=8), and provides direct easy access to individual isolated live cells for downstream single-cell manipulation and analysis. We anticipate eDAR will enable new studies of various types of rare cells that circulate in blood.

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.

Identification of genes whose expression patterns differ in benign lymphoid tissue and follicular, mantle cell, and small lymphocytic lymphoma

Improved methods for diagnosing small B-cell lymphomas (SBCLs) and predicting patient response to therapy are likely to result from the ongoing discovery of molecular markers that better define these malignancies. In this report, we identify 120 genes whose expression patterns differed between reactive lymph node tissue and three types of SBCL: follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia/small lymphocytic lymphoma. Whereas previously published studies have generally analyzed the gene expression profiles of one type of SBCL, work presented in this paper was intended to identify genes that are differentially expressed between three SBCL subtypes. This analysis was performed using mRNA pooled from multiple specimens representing each tissue type. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to validate the differential expression of 23 of these genes. Among the 23 validated genes were cyclin D1 (CCND1) and B-cell CLL/lymphoma 2, which have well-known roles in lymphoma pathogenesis. The remaining 21 genes have no currently established role in lymphoma development. Using qRT-PCR, the expression of CCND1 and seven additional genes was further studied in a panel of individual specimens. Genes identified in this study are of biological interest and represent candidate diagnostic markers.

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.Genet Med 15 12, 1004–1007.Genetics in Medicine (2013); 15 12, 1004–1007. doi:10.1038/gim.2013.51

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.Genet Med 16 10, 783–786.