The requirement for bone-marrow aspirates for genomic profiling of multiple myeloma poses an obstacle to enrolment and retention of patients in clinical trials. We evaluated whether circulating ...cell-free DNA (cfDNA) analysis is comparable to molecular profiling of myeloma using bone-marrow tumour cells. We report here a hybrid-capture-based Liquid Biopsy Sequencing (LB-Seq) method used to sequence all protein-coding exons of KRAS, NRAS, BRAF, EGFR and PIK3CA in 64 cfDNA specimens from 53 myeloma patients to >20,000 × median coverage. This method includes a variant filtering algorithm that enables detection of tumour-derived fragments present in cfDNA at allele frequencies as low as 0.25% (median 3.2%, range 0.25-46%). Using LB-Seq analysis of 48 cfDNA specimens with matched bone-marrow data, we detect 49/51 likely somatic mutations, with subclonal hierarchies reflecting tumour profiling (96% concordance), and four additional mutations likely missed by bone-marrow testing (>98% specificity). Overall, LB-Seq is a high fidelity adjunct to genetic profiling of bone-marrow in multiple myeloma.
Purpose Durable and long-term responses to the poly (ADP-ribose) polymerase inhibitor olaparib are observed in patients without BRCA1/2 mutations. However, beyond BRCA1/2 mutations, there are no ...approved biomarkers for olaparib in high-grade serous ovarian cancer (HGSOC). To determine mechanisms of durable response and resistance to olaparib therapy, we performed an analysis of HGSOC tumors from three patients without germline BRCA1/2 mutations who experienced exceptional responses to olaparib. Patients and Methods We performed integrated exome, low-pass genome, and RNA sequence analysis of tumors at diagnosis and upon relapse from patients with platinum-sensitive HGSOC recurrence who were treated > 5 years with olaparib therapy as a single agent. Results We observed somatic disruption of BRCA1/2 in all three patients at diagnosis, followed by subsequent BRCA recovery upon progression by copy number gain and/or upregulation of the remaining functional allele in two patients. The third patient with ongoing response (> 7 years) had a tumor at diagnosis with biallelic somatic deletion and loss-of-function mutation, thereby lacking a functional allele for recovery of BRCA1 activity and indicating a potential cure. Conclusion Olaparib has durable benefit for patients with ovarian cancer beyond germline BRCA1/2 carriers. These data suggest that biallelic loss of BRCA1/2 in cancer cells may be a potential marker of long-term response to poly (ADP-ribose) polymerase inhibition and that restoration of homologous repair function may be a mechanism of disease resistance.
Sustained expression of the estrogen receptor-α (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon estrogen stimulation to establish an ...oncogenic expression program. Somatic copy number alterations involving the ESR1 gene occur in approximately 1% of ESR1-positive breast cancers, suggesting that other mechanisms underlie the persistent expression of ESR1. We report significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating ESR1 in 7% of ESR1-positive breast cancers. These mutations regulate ESR1 expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by rs9383590, a functional inherited single-nucleotide variant (SNV) that accounts for several breast cancer risk-associated loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer.
Patients diagnosed with high-grade serous ovarian cancer (HGSOC) who received initial debulking surgery followed by platinum-based chemotherapy can experience highly variable clinical responses. A ...small percentage of women experience exceptional long-term survival (long term (LT), 10+ years), while others develop primary resistance to therapy and succumb to disease in less than 2 years (short term (ST)). To improve clinical management of HGSOC, there is a need to better characterize clinical and molecular profiles to identify factors that underpin these disparate survival responses.
To identify clinical and tumor molecular biomarkers associated with exceptional clinical response or resistance, we conducted an integrated clinical, exome, and transcriptome analysis of 41 primary tumors from LT (n = 20) and ST (n = 21) HGSOC patients.
Younger age at diagnosis, no residual disease post debulking surgery and low CA125 levels following surgery and chemotherapy were clinical characteristics of LT. Tumors from LT survivors had increased somatic mutation burden (median 1.62 vs. 1.22 non-synonymous mutations/Mbp), frequent BRCA1/2 biallelic inactivation through mutation and loss of heterozygosity, and enrichment of activated CD4+, CD8+ T cells, and effector memory CD4+ T cells. Characteristics of ST survival included focal copy number gain of CCNE1, lack of BRCA mutation signature, low homologous recombination deficiency scores, and the presence of ESR1-CCDC170 gene fusion.
Our findings suggest that exceptional long- or short-term survival is determined by a concert of clinical, molecular, and microenvironment factors.
Individuals and families carrying mutations in BRCA1 and BRCA2 ( BRCA1/2 ) have a markedly elevated risk of developing breast and ovarian cancers. The first-generation of BRCA1/2 mutation analysis ...targeted only the coding exons and has implicated protein-truncating mutations (indel, nonsense) in BRCA1/2 inactivation. Recently, heritable breast cancers have also been attributed to other exonic mutations (missense, silent) and mutations in introns and untranslated regions. However, analysis of these alterations has been prohibitively laborious and cost intensive, and the proportion of cases carrying mutations in unscreened regions of BRCA1/2 and other predisposition genes is unknown. We have developed and validated a next-generation sequencing (NGS) approach for BRCA1/2 mutation analysis by applying long-range PCR and deep sequencing. Genomic DNA from familial breast cancer patients ( N = 12) were screened and NGS successfully identified all 19 distinct (51 total) BRCA1 and 35 distinct (63 total) BRCA2 sequence alterations detectable by the Sanger sequencing, with no false-negative or positive results. In addition, we report the robust detection of variants from introns and untranslated regions. These results illustrate that NGS can provide comprehensive genetic information more quickly, accurately, and at a lower cost than conventional approaches, and we propose NGS to be a more effective method for BRCA1/2 mutational analysis. Advances in NGS will play an important role in enabling molecular diagnostics and personalized treatment of breast and ovarian cancers.
The genomic landscape of schwannoma Agnihotri, Sameer; Jalali, Shahrzad; Wilson, Mark R ...
Nature genetics,
11/2016, Letnik:
48, Številka:
11
Journal Article
Recenzirano
Schwannomas are common peripheral nerve sheath tumors that can cause debilitating morbidities. We performed an integrative analysis to determine genomic aberrations common to sporadic schwannomas. ...Exome sequence analysis with validation by targeted DNA sequencing of 125 samples uncovered, in addition to expected NF2 disruption, recurrent mutations in ARID1A, ARID1B and DDR1. RNA sequencing identified a recurrent in-frame SH3PXD2A-HTRA1 fusion in 12/125 (10%) cases, and genomic analysis demonstrated the mechanism as resulting from a balanced 19-Mb chromosomal inversion on chromosome 10q. The fusion was associated with male gender predominance, occurring in one out of every six men with schwannoma. Methylation profiling identified distinct molecular subgroups of schwannomas that were associated with anatomical location. Expression of the SH3PXD2A-HTRA1 fusion resulted in elevated phosphorylated ERK, increased proliferation, increased invasion and in vivo tumorigenesis. Targeting of the MEK-ERK pathway was effective in fusion-positive Schwann cells, suggesting a possible therapeutic approach for this subset of tumors.
Abstract
Background: Despite recent advances in personalized cancer therapeutics, little impact has been made on curing advanced melanomas over the last several decades. Patients with metastatic ...melanoma generally have a poor prognosis; survival is limited and typically the 5-year survival rate is less than 15% in patients with metastatic disease. Dysregulation of BRAF signaling has been shown to be one of these key drivers of the pathogenesis of disease in a large subpopulation (∼40%) of patients. Although targeted therapy with BRAF and MEK inhibitors is the mainstay of therapy for recurrent/metastatic melanomas, this treatment modality provides a temporary respite. Often resistant tumor clones(carrying mutations in oncogenes) emerge, followed by a rapid progression, causing the patients to succumb to the disease. As such, in this study, we aim to understand inter- and intra- tumor heterogeneity that leads to resistance to therapy.
Methods: We conducted deep exome sequence analysis (250X coverage) of a collection of 8 metastatic melanoma tumor samples collected by rapid autopsy from a patient who became resistant to Vemurafinib despite harboring sensitizing BRAF p. V600E mutations. We used whole exome sequence analysis to detect somatic mutations and copy number variants in every metastatic tumor. To infer subclonal populations present within each metastasis, we applied a combination of clonal inference tools to determine subclonal structures based on somatic point mutations (PyClone) and copy number alterations (TITAN). Subsequently, we applied clustering algorithms to the inferred populations to construct inter-tumor phylogenies.
Results: We found that 89% of somatic point mutations were shared across all metastatic sites and were primarily clonal. Conversely, the cellular prevalence inferred from copy number alterations showed a greater degree of subclonal evolution. We found a clear relationship between the physical locations of tumors and shared subclonal patterns, whereby metastases from lung, brain and omentum metastases clustered distinctly from those in rectum, small intestine, peritoneum, diaphragm and mesentery. To look for shared patterns of subclonal variation underlying metastatic disease and resistance to therapy, we are currently extending our analysis to an additional six patients, each with tissues from 7-9 metastatic sites collected by rapid autopsy.
Conclusion: Our analysis of multiple metastatic sites from a single patient by deep exome sequence analysis found most somatic mutations were shared across tumor sites and subclonal evolution appears to be driven primarily by acquisition of copy number alterations. Tumors in near proximity shared greater genomic similarity compare to distant sites. Ongoing analysis of additional patients is required to uncover recurrent mechanisms of resistance to targeted therapies in this disease.
Citation Format: Soroush Samadian, Prashant Bavi, Dianne Chadwick, Arnavaz Danesh, Mark Dowar, Tiantian Li, Madura Siva, Michael Roehl, Anthony m. Joshua, Trevor J. Pugh. Inferring subclonal relationships between multiple metastases from rapid autopsy of a single melanoma patient. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 167.
Abstract
Background
T-cell clonality assays (TCAs) support a variety of clinical and research interests including T-cell malignancy clone identification, minimal-residual disease (MRD) testing and ...T-cell receptor (TCR) repertoire characterization for the purposes of immunotherapy. Unfortunately, many TCAs are unable to interrogate alpha, beta, gamma, and delta TCR loci simultaneously. Aiming to overcome these limitations, we designed a T-cell clonality assay (the “NTRA”), capable of identifying T-cell gene rearrangements from all four TCR loci, using hybrid-capture followed by deep next-generation sequencing (NGS). A novel informatics package exploiting the Burrows Wheeler Alignment algorithm and finding of CDR3 seed sequences was also deployed. We then validated the assay using orthogonal methods and in a series of clinical T-cell malignancy specimens.
Methods
We used DNA probes for hybrid-capture of all V and J segments in the TCR loci, followed by NGS on the Illumina NextSeq 500 platform. Analytical validation used a series of 10 specimens, including 6 flow-cytometry characterized T-cell specimens of variable degrees of immunophenotypic uniformity and 4 cell-lines with known TCR rearrangements. PCR/gel electrophoresis and Sanger sequencing were used for orthogonal validation, using primer sets designed to test each specimen for all 90th-centile V-J configurations. Subsequently, 61 clinical T-cell lymphoma specimens were tested, each previously assessed for T-cell clonality using the clinical gold standard (i.e. PCR/ electrophoresis using the BIOMED-2 consensus primers for the TCR-beta (TRB) and TCR-gamma (TRG) loci).
Results
PCR confirmed the NTRA-identified V-J configurations with an area-under-the-curve (AUC) by receiver-operator characteristic (ROC) analysis of 0.91. Relative to single-strand Sanger sequencing, the NTRA CDR3 sequence results showed a ROC AUC of 0.83. In a DNA dilution series employing the results of a “clonal” specimen (a Jurkat cell line) spiked into a “polyclonal” specimen (a mononuclear peripheral blood specimen), we could identify “clonal” specimen-specific V-J combinations and error-corrected CDR3 sequences down to less than 10⁁-5. In the final clinical validation, the NTRA performed with a ROC AUC of 0.82 relative to the current TRB & TRG BIOMED-2 clinical assay.
Conclusions
Our novel assay can overcome the current TCR locus data-yield limitations resulting from primer-based TCAs in a single-tube. The NTRA shows comparable sensitivity and specificity relative to current standard assays and excellent performance relative to current MRD techniques when applied to clinical T-cell lymphoma specimens.
Citation Format: Etienne Mahe, David Mulder, Mark Dowar, Mahadeo Sukhai, Linh Nguyen, Pamela Ohashi, Jan Delabie, Tracy L. Stockley, Trevor Pugh, Suzanne Kamel-Reid. Improving T-cell receptor clonotyping of T-cell lymphomas using hybrid-Capture and next-generation sequencing. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 846.
Abstract
Background: Genome sequencing of multiple myeloma (MM) tumors has revealed recurrent mutations that serve as a fertile ground for targeted therapies. Indeed, activating mutations of KRAS, ...NRAS and BRAF have been reported in approximately 27%, 24% and 4% of MM cases. Based on this observation, we initiated a Phase II NCI-CTEP sponsored clinical trial of trametinib in patients with MM (PHL-9460). Detection of mutations currently require bone marrow aspirates that are invasive and can yield suboptimal samples. Targeted, ultra-deep sequencing of circulating tumor DNA (ctDNA) is a promising tool for accessing the tumor genome that has not been well studied in MM. We set out to determine the feasibility of detecting ctDNA in MM and of identifying actionable mutations and mutational load using liquid biopsy through ctDNA analysis.
Methods: MM patients enrolled onto PHL-9460 or those with heavy tumor burden were identified and consented to have their peripheral blood drawn for analysis. Where possible, matched tumor DNA was also obtained. Cell-free DNA was extracted from 7-15 mL of plasma isolated within ¬1 hour of blood draw using the QIAamp Circulating Nucleic Acid Kit and tagged with barcoded sequencing adapters for subsequent pooling. All exons of KRAS, NRAS, BRAF, PIK3CA and EGFR genes were isolated using a custom hybrid capture panel (IDT xGen Lockdown) and sequenced on an Illumina HiSeq 2000. Reads were aligned to the human genome reference (hg19) using bwa and somatic mutations were detected using muTect.
Results: We have collected 25 samples from 23 patients, 7 from patients on PHL-9460, 5 newly diagnosed, and 13 from relapsed patients having received 3.3 median prior lines of therapy (range 1-7). To date, 11 samples from 10 patients have been sequenced. The sample with the lowest DNA yield failed due to low library complexity (range 16.6-3872 ng, median yield: 197 ng). From the remaining 10 samples, the mean target coverage ranged from 31,500 to 32,500. Somatic mutations in KRAS, NRAS, or PIK3CA genes were present in 5 of 9 patients with mutant allele frequencies ranging from 1.1% to 32% (3 KRAS and 2 NRAS of which 2 cases also had a low frequency PIK3CA mutation). We did not uncover mutations in BRAF or EGFR. For patients with matched tumor DNA, mutations in ctDNA concurred with those found in tumor DNA sequencing (4 of 4 tumors with known genotypes). Two patients with NRAS mutations enrolled onto PHL-9460 have responded to trametinib (1 partial and 1 minor response) and remain on therapy.
Conclusion: ctDNA analysis in this cohort has identified key mutations in MM. The rate of RAS or RAF mutations in ctDNA compared to matched tumors and correlation to response to trametinib is ongoing and will be presented. Preliminary data suggest that ctDNA may be a reliable method of detecting mutations in MM and an alternative to bone marrow biopsy.
Citation Format: Rayan Kaedbey, Olena Kis, Arnavaz Danesh, Mark Dowar, Tiantian Li, Zhihua Li, Jessica Liu, Mark Mansour, Mahadeo Sukhai, Tong Zhang, Suzanne Kamel-Reid, Trevor J. Pugh, Suzanne Trudel. Noninvasive diagnosis of actionable mutations by deep sequencing of circulating tumor DNA in multiple myeloma. 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 615. doi:10.1158/1538-7445.AM2015-615
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