Ultra-deep Amplicon Sequencing Research Articles

Somatic variants in SLC35A2 leading to defects in N-glycosylation in mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE).

Mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE) is a new histopathological entity identified in the surgically resected brain tissue of patients with drug-resistant epilepsy. Somatic variants in SLC35A2 have been increasingly identified in MOGHE brain resections. SLC35A2 protein transports uridine 5'-diphosphogalactose (UDP-Gal) into the Golgi lumen, playing a crucial role in the process of N-glycosylation. Currently, research on the pathogenic mechanism of SLC35A2 variants in MOGHE is limited. Here we conducted genetic testing on brain samples and paired blood samples from 28 pediatric patients pathologically diagnosed with MOGHE. We performed an in-depth functional analysis of somatic variants identified in SLC35A2, integrating glycan labeling and intact glycopeptide profiling to assess N-glycosylation defects. With whole-exome sequencing and validation with ultra-deep amplicon sequencing, we identified 101 potentially pathogenic somatic variants (PPSVs) across 87 genes. Nine PPSVs in SLC35A2 were found in 10 samples. The 9 identified variants of SLC35A2, characterized by various mutation types (4 frameshift, 3 missense and 2 nonsense variants), were all confirmed to be loss-of-function via altered glycan chains. Intact glycopeptide analysis at the cellular level indicated an increase in truncated N-glycan glycoforms. Analysis of brain tissue revealed N-glycosylated proteins and glycosites modified with agalactosylated glycoforms, and glycoproteins bearing agalactosylated N-glycans were significantly enriched in cell adhesion and axon guidance-related pathways. Additionally, chemoenzymatic glycan labeling in lesions demonstrated N-glycan damage of heterotopic neurons, suggesting a potential diagnostic approach for MOGHE. Our findings provide a comprehensive somatic landscape of MOGHE and a rich resource of somatic SLC35A2 variant-related glycoform and glycoprotein abnormalities, thereby unveiling valuable insights into compromised N-glycosylation and MOGHE formation.

Somatic Mutations Detected in Parkinson Disease Could Affect Genes With a Role in Synaptic and Neuronal Processes.

The role of somatic mutations in complex diseases, including neurodevelopmental and neurodegenerative disorders, is becoming increasingly clear. However, to date, no study has shown their relation to Parkinson disease’s phenotype. To explore the relevance of embryonic somatic mutations in sporadic Parkinson disease, we performed whole-exome sequencing in blood and four brain regions of ten patients. We identified 59 candidate somatic single nucleotide variants (sSNVs) through sensitive calling and a careful filtering strategy (COSMOS). We validated 27 of them with amplicon-based ultra-deep sequencing, with a 70% validation rate for the highest-confidence variants. The identified sSNVs are in genes with synaptic functions that are co-expressed with genes previously associated with Parkinson disease. Most of the sSNVs were only called in blood but were also found in the brain tissues with ultra-deep amplicon sequencing, demonstrating the strength of multi-tissue sampling designs.

Exploring DNA Methylation Diversity in the Honey Bee Brain by Ultra-Deep Amplicon Sequencing.

Understanding methylation dynamics in organs or tissues containing many different cell types is a challenging task that cannot be efficiently addressed by the low-depth bisulphite sequencing of DNA extracted from such sources. Here we explored the feasibility of ultra-deep bisulphite sequencing of long amplicons to reveal the brain methylation patterns in three selected honey bee genes analysed across five distinct conditions on the Illumina MiSeq platform. By combing 15 libraries in one run we achieved a very high sequencing depth of 240,000–340,000 reads per amplicon, suggesting that most of the cell types in the honey bee brain, containing approximately 1 million neurons, are represented in this dataset. We found a small number of gene-specific patterns for each condition in individuals of different ages and performing distinct tasks with 80–90% of those were represented by no more than a dozen patterns. One possibility is that such a small number of frequent patterns is the result of differentially methylated epialleles, whereas the rare and less frequent patterns reflect activity-dependent modifications. The condition-specific methylation differences within each gene appear to be position-dependent with some CpGs showing significant changes and others remaining stable in a methylated or non-methylated state. Interestingly, no significant loss of methylation was detected in very old individuals. Our findings imply that these diverse patterns represent a special challenge in the analyses of DNA methylation in complex tissues and organs that cannot be investigated by low-depth genome-wide bisulphite sequencing. We conclude that ultra-deep sequencing of gene-specific amplicons combined with genotyping of differentially methylated epialleles is an effective way to facilitate more advanced neuro-epigenomic studies in honey bees and other insects.

Characterization of a Dopamine Transporter and Its Splice Variant Reveals Novel Features of Dopaminergic Regulation in the Honey Bee.

Dopamine is an important neuromodulator involved in reward-processing, movement control, motivational responses, and other aspects of behavior in most animals. In honey bees (Apis mellifera), the dopaminergic system has been implicated in an elaborate pheromonal communication network between individuals and in the differentiation of females into reproductive (queen) and sterile (worker) castes. Here we have identified and characterized a honey bee dopamine transporter (AmDAT) and a splice variant lacking exon 3 (AmDATΔex3). Both transcripts are present in the adult brain and antennae as well as at lower levels within larvae and ovaries. When expressed separately in the Xenopus oocyte system, AmDAT localizes to the oocyte surface whereas the splice variant is retained at an internal membrane. Oocytes expressing AmDAT exhibit a 12-fold increase in the uptake of [3H]dopamine relative to non-injected oocytes, whereas the AmDATΔex3-expressing oocytes show no change in [3H]dopamine transport. Electrophysiological measurements of AmDAT activity revealed it to be a high-affinity, low-capacity transporter of dopamine. The transporter also recognizes noradrenaline as a major substrate and tyramine as a minor substrate, but does not transport octopamine, L-Dopa, or serotonin. Dopamine transport via AmDAT is inhibited by cocaine in a reversible manner, but is unaffected by octopamine. Co-expression of AmDAT and AmDATΔex3 in oocytes results in a substantial reduction in AmDAT-mediated transport, which was also detected as a significant decrease in the level of AmDAT protein. This down-regulatory effect is not attributable to competition with AmDATΔex3 for ER ribosomes, nor to a general inhibition of the oocyte’s translational machinery. In vivo, the expression of both transcripts shows a high level of inter-individual variability. Gene-focused, ultra-deep amplicon sequencing detected methylation of the amdat locus at ten 5′-C-phosphate-G-3′ dinucleotides (CpGs), but only in 5–10% of all reads in whole brains or antennae. These observations, together with the localization of the amdat transcript to a few clusters of dopaminergic neurons, imply that amdat methylation is positively linked to its transcription. Our findings suggest that multiple cellular mechanisms, including gene splicing and epigenomic communication systems, may be adopted to increase the potential of a conserved gene to contribute to lineage-specific behavioral outcomes.

Ultra-deep amplicon sequencing indicates absence of low-grade mosaicism with normal cells in patients with type-1 NF1 deletions

Different types of large NF1 deletion are distinguishable by breakpoint location and potentially also by the frequency of mosaicism with normal cells lacking the deletion. However, low-grade mosaicism with fewer than 10% normal cells has not yet been excluded for all NF1 deletion types since it is impossible to assess by the standard techniques used to identify such deletions, including MLPA and array analysis. Here, we used ultra-deep amplicon sequencing to investigate the presence of normal cells in the blood of 20 patients with type-1 NF1 deletions lacking mosaicism according to MLPA. The ultra-deep sequencing entailed the screening of 96 amplicons for heterozygous SNVs located within the NF1 deletion region. DNA samples from three previously identified patients with type-2 NF1 deletions and low-grade mosaicism with normal cells as determined by FISH or microsatellite marker analysis were used to validate our methodology. In these type-2 NF1 deletion samples, proportions of 5.3%, 6.6% and 15.0% normal cells, respectively, were detected by ultra-deep amplicon sequencing. However, using this highly sensitive method, none of the 20 patients with type-1 NF1 deletions included in our analysis exhibited low-grade mosaicism with normal cells in blood, thereby supporting the view that the vast majority of type-1 deletions are germline deletions.

Detecting rare asymmetrically methylated cytosines and decoding methylation patterns in the honeybee genome.

Context-dependent gene expression in eukaryotes is controlled by several mechanisms including cytosine methylation that primarily occurs in the CG dinucleotides (CpGs). However, less frequent non-CpG asymmetric methylation has been found in various cell types, such as mammalian neurons, and recent results suggest that these sites can repress transcription independently of CpG contexts. In addition, an emerging view is that CpG hemimethylation may arise not only from deregulation of cellular processes but also be a standard feature of the methylome. Here, we have applied a novel approach to examine whether asymmetric CpG methylation is present in a sparsely methylated genome of the honeybee, a social insect with a high level of epigenetically driven phenotypic plasticity. By combining strand-specific ultra-deep amplicon sequencing of illustrator genes with whole-genome methylomics and bioinformatics, we show that rare asymmetrically methylated CpGs can be unambiguously detected in the honeybee genome. Additionally, we confirm differential methylation between two phenotypically and reproductively distinct castes, queens and workers, and offer new insight into the heterogeneity of brain methylation patterns. In particular, we challenge the assumption that symmetrical methylation levels reflect symmetry in the underlying methylation patterns and conclude that hemimethylation may occur more frequently than indicated by methylation levels. Finally, we question the validity of a prior study in which most of cytosine methylation in this species was reported to be asymmetric.

Multiple components of PKA and TGF-β pathways are mutated in pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is a subtype of mucinous adenocarcinoma mainly restricted to the peritoneal cavity and most commonly originating from the appendix. The genetic background of PMP is poorly understood and no targeted treatments are currently available for this fatal disease. While RAS signaling pathway is affected in most if not all PMP cases and over half of them also have a mutation in the GNAS gene, other genetic alterations and affected pathways are, to a large degree, poorly known. In this study, we sequenced whole coding genome of nine PMP tumors and paired normal tissues in order to identify additional, commonly mutated genes and signaling pathways affected in PMP. These exome sequencing results were validated with an ultra-deep amplicon sequencing method, leading to 14 validated variants. The validated results contain seven genes that contribute to the protein kinase A (PKA) pathway. PKA pathway, which also contains GNAS, is a major player of overproduction of mucin, which is the characteristic feature of PMP. In addition to PKA pathway, we identified mutations in six genes that belong to the transforming growth factor beta (TGF-β) pathway, which is a key regulator of cell proliferation. Since either GNAS mutation or an alternative mutation in the PKA pathway was identified in 8/9 patients, inhibition of the PKA pathway might reduce mucin production in most of the PMP patients and potentially suppress disease progression.

Abstract 5249: Intratumor heterogeneity and the detection of potential driver mutations in cfDNA in a NSCLC cohort using UltraSEEKTM

Abstract Introduction Increasing evidence supports the heterogeneous clonal composition and branched evolution of non-small cell lung cancer (NSCLC) tumors. Circulating biomarkers, such as circulating cell-free tumor DNA (cfDNA), may potentially be used to track the evolution of such tumors in order to detect minimal residual disease, monitor treatment response, detect the emergence of resistance, and subsequent disease relapse. Experimental procedures In a cohort of 14 primary NSCLC tumors, DNA was extracted from fresh frozen tissue and multi-region whole-exome sequencing (mean depth of 219x) was performed on a total of 44 tumor regions. Single nucleotide variants, indels and copy number data were identified using VarScan2 (v2.3.6). Non-silent mutations were validated using ultra-deep amplicon sequencing (mean depth 1220x). The subclonal composition of each tumor was used to construct phylogenetic trees. Candidate driver genes were selected by referencing large-scale lung cancer or pan-cancer sequencing studies, and the COSMIC gene census. To validate the identified driver mutations and to further screen all tumor regions for additional low-level heterogeneity, we developed patient-specific UltraSEEK panels. The UltraSEEK technology utilizes mutation-specific single base extension and MassARRAY® MALDI-TOF Mass Spectrometry to identify low frequency mutated alleles down to 0.1%. This method was used to determine if potential driver mutations could be detected in cfDNA extracted at the time of diagnosis in a subset of patients. Summary of data Intratumor heterogeneity was evident in all tumors, with a median of 25% heterogeneous mutations (range 5-61%). Phylogenetic tree analysis revealed branched tumor evolution, with mutations in potential driver genes occurring both early (trunk) and late (branch). The UltraSEEK method identified truncal and branch mutations in tumor DNA, as well as truncal mutations in the EGFR, TP53, BRAF, RB1 and MLL2 genes in two out of six patients. These mutations are predicted to have deleterious functional consequences and may therefore play a role in disease pathogenesis. Conclusions Spatial and temporal heterogeneity of driver mutations was evident in all NSCLC tumors. The use of liquid biopsies, such as cfDNA, has the potential to aid early detection and guide treatment initiation in cancer patients. They may also avoid the need for repeated tissue sampling, and be of value in cases where disease sites, such as brain or bone, are not easily accessible to biopsy. Citation Format: Mariam Jamal-Hanjani, Elza De Bruin, Gareth Wilson, Nicholas McGranahan, Andrew Rowan, Michael Mosko, Heath Metzler, Anders Nygren, Charles Swanton. Intratumor heterogeneity and the detection of potential driver mutations in cfDNA in a NSCLC cohort using UltraSEEKTM. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5249. doi:10.1158/1538-7445.AM2015-5249

On the identification of low allele frequency mosaic mutations in the brains of Alzheimer's disease patients

On the identification of low allele frequency mosaic mutations in the brains of Alzheimer's disease patients

Ultra-Deep Sequencing (UDS) Allows More Sensitive Detection of the D816V and Other Kit Gene Mutations in Systemic Mastocytosis

Ultra-Deep Sequencing (UDS) Allows More Sensitive Detection of the D816V and Other Kit Gene Mutations in Systemic Mastocytosis

Analysis of Clonal Evolution in Chemorefractory Acute Myeloid Leukemia from Diagnosis to Relapse

Analysis of Clonal Evolution in Chemorefractory Acute Myeloid Leukemia from Diagnosis to Relapse

Evolution of the Genomic Landscape in Non-Small Cell Lung Cancer

Evolution of the Genomic Landscape in Non-Small Cell Lung Cancer

Ultra-deep amplicon sequencing to identify actionable mutations in matched plasma/tumor specimens from 44 patients with colorectal cancer of UICC stage III and IV.

3634 Background: Circulating cell-free DNA (cf-DNA) isolated from plasma samples of cancer patients (pts) is a promising source for noninvasive examination of tumor-specific mutation patterns. We examined the efficiency of ultra-deep amplicon sequencing (UDAS) of cf-DNA isolated from plasma and tumor DNA isolated from matched primary tumor tissue of pts with colorectal cancer (CRC). Methods: Blood was drawn prior to surgery from 44 pts: 20 male, 24 female; 44-79 years of age (median: 67.5); 11 stage III, 33 stage IV; 36 colon, 8 rectum tumors; no neo-adjuvant therapy. Cf-DNA was isolated from 2 ml of EDTA plasma. UDAS was applied to 72 DNA samples (44 plasma, 28 matched snap-frozen primary tumors) using the MiSeq platform (Illumina). A panel of 49 highly multiplexed amplicons was designed (Life Technologies) representing 9 cancer genes frequently mutated in CRC. For 16 primary tumors the mutation profile was determined using the TruSeq Amplicon Cancer Panel (Illumina). Results: Median cf-DNA yield was 310 ng / 2 ml plasma (1ng – 6.600 ng). There was no significant relation between cf-DNA yield and any clinical characteristic. Median amplicon coverage was 20.162 reads per bp (2.913 - 115.782). 33/49 amplicons (67%) had a coverage of > 10.000 reads. Altogether 61 high quality COSMIC-cited mutations were confirmed in plasma of 29/44 (66%) pts: 24/33 (73%) pts in stage IV, 5/11 (45%) pts in stage III. Confirmed mutations are: APC-17; BRAF-2, FBXW7-1, KRAS-15, NRAS-1, PIK3CA-4, SMAD4-2, and TP53-19 mutations. No high quality mutations were found in CTNNB1 and HRAS. Moreover two pts exhibited four high quality plasma mutations which were not detected in the matched primary tumor: APC (R216X), KRAS (Q61K), and SMAD4 (D355E); PIK3CA (E545K). Conclusions: Ultra deep amplicon sequencing is suitable to detect mutations in plasma samples of CRC pts with a high concordance to matched primary tumors. The concordance rate can be further increased by extending the spectrum of analyzed mutations or by the enrichment of cf-DNA tumor copies. This method could be applied to detect and monitor metastasis thus opening a new paradigm for the selection of pts for targeted therapies.

Abstract 52: Next-Gen deep amplicon sequencing of TP53 complements FGFR3 for the detection of bladder cancer-related mutations.

Abstract Background: We have recently described an ultra-deep amplicon sequencing method for detection of FGFR3 mutations in urine that closely matches the frequency of mutations found in bladder cancer tissue. While FGFR3 mutations are present in a large fraction of non-invasive tumors, these mutations are rarely detected in invasive bladder tumors. To complement our existing FGFR3 deep sequencing assay, we have developed a sequencing assay for TP53. Mutations in TP53 are commonly found in advanced bladder cancer, and show little overlap with FGFR3 mutations. Here, we demonstrate the detection of FGFR3 and TP53 mutations in bladder cancer tissue using a Next-Gen sequencing platform. Methods: The FGFR3 sequencing assay was performed as described previously, permitting the detection of 9 mutations associated with bladder cancer. Similarly, amplicons were designed against TP53 exons 5-8, permitting the detection of 133 TP53 mutations previously reported in bladder cancer. Primary amplification was performed on DNA isolated from FFPE or fresh frozen tissue. The resulting PCR products were used as template for emulsion PCR and sequenced using the Ion Torrent PGM. Samples were analyzed for total DNA reads and number of mutant sequencing reads to determine percent mutation. Results: A set of 57 bladder cancer tissue samples, comprised of 38 non-muscle invasive (NMIBC) and 19 muscle invasive (MIBC) tumors, was analyzed for mutations in both FGFR3 and TP53. Of these samples, 18 out of 38 (47.4%) NMIBC tumors were positive for FGFR3 mutations, and 2 of 38 (5.3%) were positive for TP53 mutations. 9 out of 19 (47.4%) MIBC tumors were positive for TP53 mutations, with an additional 2/19 (10.9%) positive for FGFR3 mutations. Across all tumor stages tested, 35.1% (20/57) of samples were positive for FGFR3 mutations, and 19.3% (11/57) were positive for TP53 mutations. No overlap was seen between samples positive for FGFR3 or TP53 mutations. In addition, we have now shown that these assays can detect both FGFR3 and TP53 mutations in the urine of bladder cancer patients. These studies will be expanded to a large set of urine samples to more accurately assess clinical performance of this FGFR3/TP53 deep sequencing assay in bodily fluids. Conclusions: We have developed FGFR3 and TP53 sequencing assays that can detect mutations in a broad range of bladder cancer stages. The complementarity of mutations found in these two genes may allow for the non-invasive detection of bladder cancer in a larger fraction of undiagnosed patients. Citation Format: John M. Millholland, Maria C. Campo, Lydia D. Anderson, Cecilia A. Fernandez, Anthony P. Shuber. Next-Gen deep amplicon sequencing of TP53 complements FGFR3 for the detection of bladder cancer-related mutations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 52. doi:10.1158/1538-7445.AM2013-52

Combining next-gen sequencing of TP53 and FGFR3 for detecting bladder cancer-related mutations.

304 Background: We have recently described an ultra-deep amplicon sequencing method for FGFR3 in urine that closely replicates the sensitivity found in tissue. While FGFR3 mutations are present in a large fraction of non-invasive tumors, these mutations are rarely detected in invasive bladder tumors. To complement our existing FGFR3 deep sequencing assay, we have developed a sequencing assay for TP53. Mutations in TP53 are commonly found in advanced bladder cancer, and show little overlap with FGFR3 mutations. This proof of concept study demonstrates detection of FGFR3 and TP53 in bladder cancer tissue. Methods: The FGFR3 sequencing assay was performed as described previously, permitting the detection of 9 mutations associated with bladder cancer. Similarly, amplicons were designed against TP53 exons 5-8 using, permitting the detection of 133 unique TP53 mutations previously detected in bladder cancer. Primary amplification was performed on DNA isolated from 3 10µ tissue sections. The resulting PCR products were used as template for emulsion PCR and sequenced using the Ion Torrent PGM. Samples were analyzed for total DNA reads and number of mutant sequencing reads to determine percent mutation. Results: Previously we analyzed 151 non-invasive bladder tumor samples for the presence of FGFR3 mutations. Of these samples, 93 out of 151 (61.5%) were positive for FGFR3 mutations. An additional set of 10 high stage bladder tumor samples were analyzed for mutations in TP53. Of these samples, 5 out of 10 (50.0%) were positive for TP53 mutations, while a separate sample was found to be positive for FGFR3 only. Importantly, a TP53 mutation was detected in a Tis carcinoma in situ sample. These studies will be expanded to a larger set of bladder cancer tissues and urine samples to more accurately assess clinical performance of this FGFR3/TP53 deep sequencing assay. Conclusions: We have developed a multiplexed FGFR3 and TP53 sequencing assay that can detect mutations in a broad range of bladder cancer stages. The complementarity of mutations found in these two genes may allow for the detection of a larger fraction of patients with undiagnosed bladder cancer.

Ultradeep sequencing detects GNAQ and GNA11 mutations in cell‐free DNA from plasma of patients with uveal melanoma

Elevated levels of cell-free DNA (cfDNA) are frequently observed in tumor patients. Activating mutations in exon 4 (R183) and exon 5 (Q209) of GNAQ and GNA11 are almost exclusively found in uveal melanoma, thus providing a highly specific marker for the presence of circulating tumor DNA (ctDNA). To establish a reliable, noninvasive assay that might allow early detection and monitoring of metastatic disease, we determined the proportion of GNAQ or GNA11 mutant reads in cfDNA of uveal melanoma patients by ultradeep sequencing. Cell-free DNA from 28 uveal melanoma patients with metastases or extraocular growth was isolated and quantified by real-time polymerase chain reaction (PCR) (7–1550 ng DNA/mL plasma). GNAQ and GNA11 regions of interest were amplified in 22 of 28 patients and ultradeep sequencing of amplicons was performed to detect even low proportions of mutant reads. We detected Q209 mutations (2–38% mutant reads) in either GNAQ or GNA11 in the plasma of 9 of 22 metastasized patients. No correlation between the proportion of mutant reads and the concentration of cfDNA could be detected. Among the nine ctDNA-positive patients, four had metastases in bone, whereas no metastases were detected in the 13 ctDNA-negative patients at this location (P = 0.025). Furthermore, ctDNA-positive patients tended to be younger at initial diagnosis and show larger metastases. The results show that ultradeep amplicon sequencing can be used to detect tumor DNA in plasma of metastasized uveal melanoma patients. It remains to be shown if this approach can be used for early detection of disseminated tumor disease.

Ultradeep-Amplicon Pyrosequencing for Mutation Detection in the Kinase Domain of BCR-ABL Revealed Artificial Low-Level Variants That Need to Be Avoided for Relevant Mutational Data Interpretation

Ultradeep-Amplicon Pyrosequencing for Mutation Detection in the Kinase Domain of BCR-ABL Revealed Artificial Low-Level Variants That Need to Be Avoided for Relevant Mutational Data Interpretation

Detection of low-frequency FGFR3 mutations in the urine of patients with bladder cancer using next-generation deep sequencing.

59 Background: FGFR3 mutations have been identified in ~60-70% of low-stage, non-invasive tumors. Our group and others have developed assays to detect FGFR3 mutations in the urine of bladder cancer patients. However, urine-based assays have been limited by the technical ability to detect rare events in a dilute medium where there is a high background of normal DNA. In these assays, FGFR3 mutations are generally found in ~30% of the urine samples, which is < 50% concordance with the expected detection in tissue. We have now developed an ultra-deep amplicon sequencing technique that increases FGFR3 mutation detection in urine to ~67%, close to the expected detection frequency if every mutation found in tissue could be detected in urine. Methods: Amplicons were designed against FGFR3 exons 7, 10, and 15 using PCR primers containing the adapter sequences for unidirectional sequencing. Primary amplification was performed from DNA isolated from 4 ml of urine. The resulting PCR products were used as template for emulsion PCR and these were then sequenced using the Roche 454 GS Junior. Samples were analyzed for total DNA reads per sample and number of mutant sequencing reads to determine percent mutation. Results: Urine samples from 43 patients with bladder cancer were analyzed by both our previously described qPCR method and the new ultra-deep sequencing approach. Using ultra-deep amplicon sequencing, 24 out of 43 (55.8%) were positive for FGFR3 mutations, while only 5 out of 43 (11.6%) were positive for mutations by qPCR. The urine samples from the 15 newly identified mutations using deep sequencing contained FGFR3 mutations as low as 0.05% mutant DNA. The sensitivity achieved using deep sequencing was 91% concordant with the FGFR3 mutations observed in tissue. Conclusions: We have developed a highly sensitive non-invasive urine based assay that can detect FGFR3 mutant DNA when present at < 1% of the sample and is > 90% concordance with the mutations found in tumor tissues. To our knowledge, this is the first practical application of next generation sequencing technology for diagnostic use.

Abstract 2093: Next-gen deep sequencing improves FGFR3 mutation detection in the urine of bladder cancer patients

Abstract FGFR3 mutations have been identified in ∼60-70% of low-stage, non-invasive tumors. Our group and others have developed assays to detect FGFR3 mutations in the urine of bladder cancer patients. However, urine-based assays have been limited by the technical ability to detect rare events in a dilute medium where there is a high background of normal DNA. In these assays, FGFR3 mutations are generally found in ∼30% of the urine samples, which is < 50% concordance with the expected detection in tissue. We have now developed an ultra-deep amplicon sequencing technique that increases FGFR3 mutation detection in urine to ∼67%, near the expected detection if every mutation found in tissue could be detected in urine. Amplicons were designed against FGFR3 exons 7, 10, and 15 using PCR primers containing the adapter sequences for unidirectional sequencing. Taqman probes were used to determine if sufficient DNA was present in each sample. Primary amplification was performed from DNA isolated from 4 ml of urine. The resulting PCR products were used as template for emulsion PCR and these were then sequenced using the Roche 454 GS Junior. Samples were analyzed for total DNA reads per sample and number of mutant sequencing reads to determine percent mutation. Urine samples from 29 patients with stage Ta bladder cancer were analyzed by both our previously described qPCR method and the new ultra-deep sequencing approach. Of the 29 samples, 2 did not have sufficient DNA for analysis by sequencing. Using ultra-deep amplicon sequencing, 18 out of 27 (66.7%) were positive for FGFR3 mutations, while only 3 out of 27 (11.1%) were positive for mutations by qPCR. The urine samples from the 15 newly identified mutations using deep sequencing contained FGFR3 mutations as low as 0.05%. Using matched tissue and urine samples, we demonstrate that the sensitivity in urine approximates the FGFR3 mutations observed in tissue. We have developed a highly sensitive non-invasive urine based assay that can detect FGFR3 mutant DNA when present at < 1% of the sample and that achieves > 90% concordance with tumor tissues. To our knowledge, this is the first practical application of next generation sequencing technology for diagnostic use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2093. doi:1538-7445.AM2012-2093

Abstract 1884: Resistance to second-line tyrosine kinase inhibitor treatment in imatinib-resistant Philadelphia-positive leukemia patients can be anticipated by ultra-deep sequencing of the BCR-ABL kinase domain using Roche 454 technology

Abstract Definite spectra of point mutations in the Bcr-Abl kinase domain (KD) are known to confer resistance to the 1st-generation tyrosine kinase inhibitor (TKI) imatinib (IM) and to the 2nd-generation TKIs dasatinib (DAS) and nilotinib (NIL) in Philadelphia-positive (Ph+) leukemias. Currently, European LeukemiaNet guidelines recommend mutation analysis to be performed by denaturing-high performance liquid chromatography (D-HPLC) and/or direct sequencing. Using Roche 454 next-generation sequencing (NGS) technology on a GS Junior instrument, we investigated whether an ultra-deep amplicon sequencing strategy for mutation screening of the Bcr-Abl KD could highlight emerging clones harbouring critical mutations, ultimately anticipating the development of drug-resistance. A lower detection limit of 0.05% - 5 mutated Bcr-Abl transcripts in a total of 10,000 Bcr-Abl transcripts - could routinely be achieved. Serially collected samples from IM-resistant CML and Ph+ ALL pts who received DAS and/or NIL as 2nd- or 3rd-line therapy and developed DAS- or NIL-resistant mutations were retrospectively analyzed from the time of switchover to the time of relapse. NGS was able to confirm all mutations known to be present in >20% of cells, allowed to follow quantitatively the dynamics of mutated clones over time and showed that i) 9/12 IM-resistant pts who failed to achieve a satisfactory response to 2nd-line therapy (primary resistance) were already harbouring mutations known to be insensitive to DAS (T315I, F317L/V) or NIL (E255K/V, Y253H, F359V/C) at 1-10% level before the TKI start - despite they had been scored as wild-type by D-HPLC; ii) in 9/13 IM-resistant pts who relapsed after an initial response (acquired resistance), the emerging resistant clone(s) carrying DAS- or NIL-insensitive mutation(s) could be identified up to six month in advance than when D-HPLC and conventional sequencing were used. In addition, when used for mutation monitoring of newly diagnosed Ph+ ALL pts enrolled in clinical trials with DAS or NIL, NGS again highlighted emerging resistant clones in 7/8 pts who later relapsed. We conclude that the higher sensitivity of NGS allows to detect emerging mutant clones earlier than the currently used methods; this is particularly relevant: i) in IM-resistant CML and Ph+ ALL pts before switching to and during treatment with a 2nd-line TKI, where NGS may anticipate the presence of DAS- or NIL-insensitive mutations, ensuring the most effective and timely therapeutic decisions; ii) more in general, for mutation monitoring of Ph+ ALL pts, that are known to be highly genetically unstable, hence markedly prone to develop resistance and mutations while on TKI therapy. Based on our data, further evaluation of the routine applicability of NGS is warranted. Supported by AIL, AIRC, PRIN, ELN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1884. doi:1538-7445.AM2012-1884