Publications by authors named "Kilannin Krysiak"

30 Publications

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Corrigendum to "Haploinsufficiency of multiple del(5q) genes induce B cell abnormalities in mice" [Leuk. Res. 96C (2020) 106428].

Leuk Res 2021 Jan 4;100:106478. Epub 2020 Dec 4.

Department of Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, United States; Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO, United States. Electronic address:

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http://dx.doi.org/10.1016/j.leukres.2020.106478DOI Listing
January 2021

Haploinsufficiency of multiple del(5q) genes induce B cell abnormalities in mice.

Leuk Res 2020 09 23;96:106428. Epub 2020 Jul 23.

Department of Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, Missouri, United States; Siteman Cancer Center, Washington University School of Medicine, St. Louis, Missouri, United States. Electronic address:

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http://dx.doi.org/10.1016/j.leukres.2020.106428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768807PMC
September 2020

A harmonized meta-knowledgebase of clinical interpretations of somatic genomic variants in cancer.

Nat Genet 2020 04 3;52(4):448-457. Epub 2020 Apr 3.

MolecularMatch, Houston, TX, USA.

Precision oncology relies on accurate discovery and interpretation of genomic variants, enabling individualized diagnosis, prognosis and therapy selection. We found that six prominent somatic cancer variant knowledgebases were highly disparate in content, structure and supporting primary literature, impeding consensus when evaluating variants and their relevance in a clinical setting. We developed a framework for harmonizing variant interpretations to produce a meta-knowledgebase of 12,856 aggregate interpretations. We demonstrated large gains in overlap between resources across variants, diseases and drugs as a result of this harmonization. We subsequently demonstrated improved matching between a patient cohort and harmonized interpretations of potential clinical significance, observing an increase from an average of 33% per individual knowledgebase to 57% in aggregate. Our analyses illuminate the need for open, interoperable sharing of variant interpretation data. We also provide a freely available web interface (search.cancervariants.org) for exploring the harmonized interpretations from these six knowledgebases.
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http://dx.doi.org/10.1038/s41588-020-0603-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127986PMC
April 2020

CIViCpy: A Python Software Development and Analysis Toolkit for the CIViC Knowledgebase.

JCO Clin Cancer Inform 2020 03;4:245-253

McDonnell Genome Institute, Washington University School of Medicine, St Louis, MO.

Purpose: Precision oncology depends on the matching of tumor variants to relevant knowledge describing the clinical significance of those variants. We recently developed the Clinical Interpretations for Variants in Cancer (CIViC; civicdb.org) crowd-sourced, expert-moderated, and open-access knowledgebase. CIViC provides a structured framework for evaluating genomic variants of various types (eg, fusions, single-nucleotide variants) for their therapeutic, prognostic, predisposing, diagnostic, or functional utility. CIViC has a documented application programming interface for accessing CIViC records: assertions, evidence, variants, and genes. Third-party tools that analyze or access the contents of this knowledgebase programmatically must leverage this application programming interface, often reimplementing redundant functionality in the pursuit of common analysis tasks that are beyond the scope of the CIViC Web application.

Methods: To address this limitation, we developed CIViCpy (civicpy.org), a software development kit for extracting and analyzing the contents of the CIViC knowledgebase. CIViCpy enables users to query CIViC content as dynamic objects in Python. We assess the viability of CIViCpy as a tool for advancing individualized patient care by using it to systematically match CIViC evidence to observed variants in patient cancer samples.

Results: We used CIViCpy to evaluate variants from 59,437 sequenced tumors of the American Association for Cancer Research Project GENIE data set. We demonstrate that CIViCpy enables annotation of > 1,200 variants per second, resulting in precise variant matches to CIViC level A (professional guideline) or B (clinical trial) evidence for 38.6% of tumors.

Conclusion: The clinical interpretation of genomic variants in cancers requires high-throughput tools for interoperability and analysis of variant interpretation knowledge. These needs are met by CIViCpy, a software development kit for downstream applications and rapid analysis. CIViCpy is fully documented, open-source, and available free online.
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http://dx.doi.org/10.1200/CCI.19.00127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113080PMC
March 2020

Text-mining clinically relevant cancer biomarkers for curation into the CIViC database.

Genome Med 2019 12 3;11(1):78. Epub 2019 Dec 3.

Canada's Michael Smith Genome Sciences Centre, Vancouver, BC, Canada.

Background: Precision oncology involves analysis of individual cancer samples to understand the genes and pathways involved in the development and progression of a cancer. To improve patient care, knowledge of diagnostic, prognostic, predisposing, and drug response markers is essential. Several knowledgebases have been created by different groups to collate evidence for these associations. These include the open-access Clinical Interpretation of Variants in Cancer (CIViC) knowledgebase. These databases rely on time-consuming manual curation from skilled experts who read and interpret the relevant biomedical literature.

Methods: To aid in this curation and provide the greatest coverage for these databases, particularly CIViC, we propose the use of text mining approaches to extract these clinically relevant biomarkers from all available published literature. To this end, a group of cancer genomics experts annotated sentences that discussed biomarkers with their clinical associations and achieved good inter-annotator agreement. We then used a supervised learning approach to construct the CIViCmine knowledgebase.

Results: We extracted 121,589 relevant sentences from PubMed abstracts and PubMed Central Open Access full-text papers. CIViCmine contains over 87,412 biomarkers associated with 8035 genes, 337 drugs, and 572 cancer types, representing 25,818 abstracts and 39,795 full-text publications.

Conclusions: Through integration with CIVIC, we provide a prioritized list of curatable clinically relevant cancer biomarkers as well as a resource that is valuable to other knowledgebases and precision cancer analysts in general. All data is publically available and distributed with a Creative Commons Zero license. The CIViCmine knowledgebase is available at http://bionlp.bcgsc.ca/civicmine/.
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http://dx.doi.org/10.1186/s13073-019-0686-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891984PMC
December 2019

Standard operating procedure for curation and clinical interpretation of variants in cancer.

Genome Med 2019 11 29;11(1):76. Epub 2019 Nov 29.

McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO, USA.

Manually curated variant knowledgebases and their associated knowledge models are serving an increasingly important role in distributing and interpreting variants in cancer. These knowledgebases vary in their level of public accessibility, and the complexity of the models used to capture clinical knowledge. CIViC (Clinical Interpretation of Variants in Cancer - www.civicdb.org) is a fully open, free-to-use cancer variant interpretation knowledgebase that incorporates highly detailed curation of evidence obtained from peer-reviewed publications and meeting abstracts, and currently holds over 6300 Evidence Items for over 2300 variants derived from over 400 genes. CIViC has seen increased adoption by, and also undertaken collaboration with, a wide range of users and organizations involved in research. To enhance CIViC's clinical value, regular submission to the ClinVar database and pursuit of other regulatory approvals is necessary. For this reason, a formal peer reviewed curation guideline and discussion of the underlying principles of curation is needed. We present here the CIViC knowledge model, standard operating procedures (SOP) for variant curation, and detailed examples to support community-driven curation of cancer variants.
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http://dx.doi.org/10.1186/s13073-019-0687-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883603PMC
November 2019

Open-Sourced CIViC Annotation Pipeline to Identify and Annotate Clinically Relevant Variants Using Single-Molecule Molecular Inversion Probes.

JCO Clin Cancer Inform 2019 10;3:1-12

Washington University School of Medicine, St Louis, MO.

Purpose: Clinical targeted sequencing panels are important for identifying actionable variants for patients with cancer; however, existing approaches do not provide transparent and rationally designed clinical panels to accommodate the rapidly growing knowledge within oncology.

Materials And Methods: We used the Clinical Interpretations of Variants in Cancer (CIViC) database to develop an Open-Sourced CIViC Annotation Pipeline (OpenCAP). OpenCAP provides methods to identify variants within the CIViC database, build probes for variant capture, use probes on prospective samples, and link somatic variants to CIViC clinical relevance statements. OpenCAP was tested using a single-molecule molecular inversion probe (smMIP) capture design on 27 cancer samples from 5 tumor types. In total, 2,027 smMIPs were designed to target 111 eligible CIViC variants (61.5 kb of genomic space).

Results: When compared with orthogonal sequencing, CIViC smMIP sequencing demonstrated a 95% sensitivity for variant detection (n = 61 of 64 variants). Variant allele frequencies for variants identified on both sequencing platforms were highly concordant (Pearson's = 0.885; n = 61 variants). Moreover, for individuals with paired tumor and normal samples (n = 12), 182 clinically relevant variants missed by orthogonal sequencing were discovered by CIViC smMIP sequencing.

Conclusion: The OpenCAP design paradigm demonstrates the utility of an open-source and open-access database built on attendant community contributions with peer-reviewed interpretations. Use of a public repository for variant identification, probe development, and variant interpretation provides a transparent approach to build dynamic next-generation sequencing-based oncology panels.
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http://dx.doi.org/10.1200/CCI.19.00077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873961PMC
October 2019

A Spontaneous Aggressive ERα+ Mammary Tumor Model Is Driven by Kras Activation.

Cell Rep 2019 08;28(6):1526-1537.e4

McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA; Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63108, USA; Department of Genetics, Washington University School of Medicine, St. Louis, MO 63108, USA; Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO 63108, USA. Electronic address:

The NRL-PRL murine model, defined by mammary-selective transgenic rat prolactin ligand rPrl expression, establishes spontaneous ER+ mammary tumors in nulliparous females, mimicking the association between elevated prolactin (PRL) and risk for development of ER+ breast cancer in postmenopausal women. Whole-genome and exome sequencing in a discovery cohort (n = 5) of end-stage tumors revealed canonical activating mutations and copy number amplifications of Kras. The frequent mutations in this pathway were validated in an extension cohort, identifying activating Ras alterations in 79% of tumors (23 of 29). Transcriptome analyses over the course of oncogenesis revealed marked alterations associated with Ras activity in established tumors compared with preneoplastic tissues; in cell-intrinsic processes associated with mitosis, cell adhesion, and invasion; as well as in the surrounding tumor environment. These genomic analyses suggest that PRL induces a selective bottleneck for spontaneous Ras-driven tumors that may model a subset of aggressive clinical ER+ breast cancers.
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http://dx.doi.org/10.1016/j.celrep.2019.06.098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713291PMC
August 2019

Author Correction: The prognostic effects of somatic mutations in ER-positive breast cancer.

Nat Commun 2018 11 14;9(1):4850. Epub 2018 Nov 14.

McDonnell Genome Institute, Washington University School of Medicine, St. Louis 63108, MO, USA.

The original version of this Article contained errors in the depiction of confidence intervals in the NF1 BCSS data illustrated in Figure 3b. These have now been corrected in both the PDF and HTML versions of the Article. The incorrect version of Figure 3b is presented in the associated Author Correction.
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http://dx.doi.org/10.1038/s41467-018-07407-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235964PMC
November 2018

Adapting crowdsourced clinical cancer curation in CIViC to the ClinGen minimum variant level data community-driven standards.

Hum Mutat 2018 11;39(11):1721-1732

McDonnell Genome Institute, Washington University School of Medicine, Saint Louis, Missouri.

Harmonization of cancer variant representation, efficient communication, and free distribution of clinical variant-associated knowledge are central problems that arise with increased usage of clinical next-generation sequencing. The Clinical Genome Resource (ClinGen) Somatic Working Group (WG) developed a minimal variant level data (MVLD) representation of cancer variants, and has an ongoing collaboration with Clinical Interpretations of Variants in Cancer (CIViC), an open-source platform supporting crowdsourced and expert-moderated cancer variant curation. Harmonization between MVLD and CIViC variant formats was assessed by formal field-by-field analysis. Adjustments to the CIViC format were made to harmonize with MVLD and support ClinGen Somatic WG curation activities, including four new features in CIViC: (1) introduction of an assertions feature for clinical variant assessment following the Association of Molecular Pathologists (AMP) guidelines, (2) group-level curation tracking for organizations, enabling member transparency, and curation effort summaries, (3) introduction of ClinGen Allele Registry IDs to CIViC, and (4) mapping of CIViC assertions into ClinVar submission with automated submissions. A generalizable workflow utilizing MVLD and new CIViC features is outlined for use by ClinGen Somatic WG task teams for curation and submission to ClinVar, and provides a model for promoting harmonization of cancer variant representation and efficient distribution of this information.
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http://dx.doi.org/10.1002/humu.23651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282863PMC
November 2018

Standard operating procedure for somatic variant refinement of sequencing data with paired tumor and normal samples.

Genet Med 2019 04 5;21(4):972-981. Epub 2018 Oct 5.

McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO, USA.

Purpose: Following automated variant calling, manual review of aligned read sequences is required to identify a high-quality list of somatic variants. Despite widespread use in analyzing sequence data, methods to standardize manual review have not been described, resulting in high inter- and intralab variability.

Methods: This manual review standard operating procedure (SOP) consists of methods to annotate variants with four different calls and 19 tags. The calls indicate a reviewer's confidence in each variant and the tags indicate commonly observed sequencing patterns and artifacts that inform the manual review call. Four individuals were asked to classify variants prior to, and after, reading the SOP and accuracy was assessed by comparing reviewer calls with orthogonal validation sequencing.

Results: After reading the SOP, average accuracy in somatic variant identification increased by 16.7% (p value = 0.0298) and average interreviewer agreement increased by 12.7% (p value < 0.001). Manual review conducted after reading the SOP did not significantly increase reviewer time.

Conclusion: This SOP supports and enhances manual somatic variant detection by improving reviewer accuracy while reducing the interreviewer variability for variant calling and annotation.
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http://dx.doi.org/10.1038/s41436-018-0278-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450397PMC
April 2019

Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer.

Nat Commun 2018 09 17;9(1):3787. Epub 2018 Sep 17.

Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110, USA.

Nearly all patients with small cell lung cancer (SCLC) eventually relapse with chemoresistant disease. The molecular mechanisms driving chemoresistance in SCLC remain un-characterized. Here, we describe whole-exome sequencing of paired SCLC tumor samples procured at diagnosis and relapse from 12 patients, and unpaired relapse samples from 18 additional patients. Multiple somatic copy number alterations, including gains in ABCC1 and deletions in MYCL, MSH2, and MSH6, are identifiable in relapsed samples. Relapse samples also exhibit recurrent mutations and loss of heterozygosity in regulators of WNT signaling, including CHD8 and APC. Analysis of RNA-sequencing data shows enrichment for an ASCL1-low expression subtype and WNT activation in relapse samples. Activation of WNT signaling in chemosensitive human SCLC cell lines through APC knockdown induces chemoresistance. Additionally, in vitro-derived chemoresistant cell lines demonstrate increased WNT activity. Overall, our results suggest WNT signaling activation as a mechanism of chemoresistance in relapsed SCLC.
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http://dx.doi.org/10.1038/s41467-018-06162-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141466PMC
September 2018

The prognostic effects of somatic mutations in ER-positive breast cancer.

Nat Commun 2018 09 4;9(1):3476. Epub 2018 Sep 4.

McDonnell Genome Institute, Washington University School of Medicine, St. Louis, 63108, MO, USA.

Here we report targeted sequencing of 83 genes using DNA from primary breast cancer samples from 625 postmenopausal (UBC-TAM series) and 328 premenopausal (MA12 trial) hormone receptor-positive (HR+) patients to determine interactions between somatic mutation and prognosis. Independent validation of prognostic interactions was achieved using data from the METABRIC study. Previously established associations between MAP3K1 and PIK3CA mutations with luminal A status/favorable prognosis and TP53 mutations with Luminal B/non-luminal tumors/poor prognosis were observed, validating the methodological approach. In UBC-TAM, NF1 frame-shift nonsense (FS/NS) mutations were also a poor outcome driver that was validated in METABRIC. For MA12, poor outcome associated with PIK3R1 mutation was also reproducible. DDR1 mutations were strongly associated with poor prognosis in UBC-TAM despite stringent false discovery correction (q = 0.0003). In conclusion, uncommon recurrent somatic mutations should be further explored to create a more complete explanation of the highly variable outcomes that typifies ER+ breast cancer.
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http://dx.doi.org/10.1038/s41467-018-05914-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123466PMC
September 2018

Single-agent ibrutinib in relapsed or refractory follicular lymphoma: a phase 2 consortium trial.

Blood 2018 01 26;131(2):182-190. Epub 2017 Oct 26.

Division of Oncology and Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO.

Most patients with follicular lymphoma (FL) experience multiple relapses necessitating subsequent lines of therapy. Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor approved for the treatment of several B-cell malignancies, showed promising activity in FL in a phase 1 study. We report the results of a phase 2 trial evaluating ibrutinib in recurrent FL. Forty patients with recurrent FL were treated with ibrutinib 560 mg/d until progression or intolerance. The primary end point was overall response rate (ORR). Exploratory analyses included correlations of outcome with recurrent mutations identified in a cancer gene panel that used next-generation sequencing in pretreatment biopsies from 31 patients and results of early interim positron emission tomography/computed tomography scans in 20 patients. ORR was 37.5% with a complete response rate of 12.5%, median progression-free survival (PFS) of 14 months, and 2-year PFS of 20.4%. Response rates were significantly higher among patients whose disease was sensitive to rituximab (52.6%) compared with those who were rituximab refractory (16.7%) ( = .04). CARD11 mutations were present in 16% of patients (5 of 31) and predicted resistance to ibrutinib with only wild-type patients responding ( = .002). Maximum standardized uptake value at cycle 1 day 8 correlated with response and PFS. Ibrutinib was well-tolerated with a toxicity profile similar to labeled indications. Ibrutinib is a well-tolerated treatment with modest activity in relapsed FL. Evaluation of BTK inhibitors in earlier lines of therapy may be warranted on the basis of improved response rates in rituximab-sensitive disease. Somatic mutations such as may have an impact on response to ibrutinib, may inform clinical decisions, and should be evaluated in larger data sets. This trial was registered at www.clinicaltrials.gov as #NCT01849263.
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http://dx.doi.org/10.1182/blood-2017-09-804641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757691PMC
January 2018

A Phase II Trial of Neoadjuvant MK-2206, an AKT Inhibitor, with Anastrozole in Clinical Stage II or III -Mutant ER-Positive and HER2-Negative Breast Cancer.

Clin Cancer Res 2017 Nov 5;23(22):6823-6832. Epub 2017 Sep 5.

Department of Medicine, Washington University School of Medicine, St. Louis, Missouri.

Hyperactivation of AKT is common and associated with endocrine resistance in estrogen receptor-positive (ER) breast cancer. The allosteric pan-AKT inhibitor MK-2206 induced apoptosis in -mutant ER breast cancer under estrogen-deprived condition in preclinical studies. This neoadjuvant phase II trial was therefore conducted to test the hypothesis that adding MK-2206 to anastrozole induces pathologic complete response (pCR) in mutant ER breast cancer. Potential eligible patients with clinical stage II/III ER/HER2 breast cancer were preregistered and received anastrozole (goserelin if premenopausal) for 28 days in cycle 0 pending tumor sequencing. Patients positive for mutation in the tumor were eligible to start MK-2206 (150 mg orally weekly, with prophylactic prednisone) on cycle 1 day 2 (C1D2) and to receive a maximum of four 28-day cycles of combination therapy before surgery. Serial biopsies were collected at preregistration, C1D1 and C1D17. Fifty-one patients preregistered and 16 of 22 with -mutant tumors received study drug. Three patients went off study due to C1D17 Ki67 >10% ( = 2) and toxicity ( = 1). Thirteen patients completed neoadjuvant therapy followed by surgery. No pCRs were observed. Rash was common. MK-2206 did not further suppress cell proliferation and did not induce apoptosis on C1D17 biopsies. Although AKT phosphorylation was reduced, PRAS40 phosphorylation at C1D17 after MK-2206 persisted. One patient acquired an mutation at surgery. MK-2206 is unlikely to add to the efficacy of anastrozole alone in -mutant ER breast cancer and should not be studied further in the target patient population. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-1260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392430PMC
November 2017

Melorheostosis: Exome sequencing of an associated dermatosis implicates postzygotic mosaicism of mutated KRAS.

Bone 2017 Aug 21;101:145-155. Epub 2017 Apr 21.

McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA; Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO 63110, USA; Division of Genomics and Bioinformatics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Melorheostosis (MEL) is the rare sporadic dysostosis characterized by monostotic or polyostotic osteosclerosis and hyperostosis often distributed in a sclerotomal pattern. The prevailing hypothesis for MEL invokes postzygotic mosaicism. Sometimes scleroderma-like skin changes, considered a representation of the pathogenetic process of MEL, overlie the bony changes, and sometimes MEL becomes malignant. Osteopoikilosis (OPK) is the autosomal dominant skeletal dysplasia that features symmetrically distributed punctate osteosclerosis due to heterozygous loss-of-function mutation within LEMD3. Rarely, radiographic findings of MEL occur in OPK. However, germline mutation of LEMD3 does not explain sporadic MEL. To explore if mosaicism underlies MEL, we studied a boy with polyostotic MEL and characteristic overlying scleroderma-like skin, a few bony lesions consistent with OPK, and a large epidermal nevus known to usually harbor a HRAS, FGFR3, or PIK3CA gene mutation. Exome sequencing was performed to ~100× average read depth for his two dermatoses, two areas of normal skin, and peripheral blood leukocytes. As expected for non-malignant tissues, the patient's mutation burden in his normal skin and leukocytes was low. He, his mother, and his maternal grandfather carried a heterozygous, germline, in-frame, 24-base-pair deletion in LEMD3. Radiographs of the patient and his mother revealed bony foci consistent with OPK, but she showed no MEL. For the patient, somatic variant analysis, using four algorithms to compare all 20 possible pairwise combinations of his five DNA samples, identified only one high-confidence mutation, heterozygous KRAS Q61H (NM_033360.3:c.183A>C, NP_203524.1:p.Gln61His), in both his dermatoses but absent in his normal skin and blood. Thus, sparing our patient biopsy of his MEL bone, we identified a heterozygous somatic KRAS mutation in his scleroderma-like dermatosis considered a surrogate for MEL. This implicates postzygotic mosaicism of mutated KRAS, perhaps facilitated by germline LEMD3 haploinsufficiency, causing his MEL.
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http://dx.doi.org/10.1016/j.bone.2017.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518630PMC
August 2017

Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells.

PLoS One 2017 8;12(2):e0170470. Epub 2017 Feb 8.

Department of Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

Myelodysplastic syndromes (MDS) are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q) cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q) cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q) compared to non-del(5q) MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170470PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5298293PMC
August 2017

Recurrent somatic mutations affecting B-cell receptor signaling pathway genes in follicular lymphoma.

Blood 2017 01 14;129(4):473-483. Epub 2016 Nov 14.

McDonnell Genome Institute, Department of Medicine.

Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma, yet it remains only partially characterized at the genomic level. To improve our understanding of the genetic underpinnings of this incurable and clinically heterogeneous disease, whole-exome sequencing was performed on tumor/normal pairs from a discovery cohort of 24 patients with FL. Using these data and mutations identified in other B-cell malignancies, 1716 genes were sequenced in 113 FL tumor samples from 105 primarily treatment-naive individuals. We identified 39 genes that were mutated significantly above background mutation rates. CREBBP mutations were associated with inferior PFS. In contrast, mutations in previously unreported HVCN1, a voltage-gated proton channel-encoding gene and B-cell receptor signaling modulator, were associated with improved PFS. In total, 47 (44.8%) patients harbor mutations in the interconnected B-cell receptor (BCR) and CXCR4 signaling pathways. Histone gene mutations were more frequent than previously reported (identified in 43.8% of patients) and often co-occurred (17.1% of patients). A novel, recurrent hotspot was identified at a posttranslationally modified residue in the histone H2B family. This study expands the number of mutated genes described in several known signaling pathways and complexes involved in lymphoma pathogenesis (BCR, Notch, SWitch/sucrose nonfermentable (SWI/SNF), vacuolar ATPases) and identified novel recurrent mutations (EGR1/2, POU2AF1, BTK, ZNF608, HVCN1) that require further investigation in the context of FL biology, prognosis, and treatment.
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http://dx.doi.org/10.1182/blood-2016-07-729954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270390PMC
January 2017

Truncating Prolactin Receptor Mutations Promote Tumor Growth in Murine Estrogen Receptor-Alpha Mammary Carcinomas.

Cell Rep 2016 09;17(1):249-260

McDonnell Genome Institute, Washington University School of Medicine, 4444 Forest Park Ave., St. Louis, MO 63108, USA; Department of Medicine, Washington University School of Medicine, 660 S Euclid Ave., St. Louis, MO 63110, USA; Siteman Cancer Center, Washington University School of Medicine, 4921 Parkview Pl., St. Louis, MO 63110, USA; Department of Genetics, Washington University School of Medicine, 660 S Euclid Ave., St. Louis, MO 63110, USA. Electronic address:

Estrogen receptor alpha-positive (ERα+) luminal tumors are the most frequent subtype of breast cancer. Stat1(-/-) mice develop mammary tumors that closely recapitulate the biological characteristics of this cancer subtype. To identify transforming events that contribute to tumorigenesis, we performed whole genome sequencing of Stat1(-/-) primary mammary tumors and matched normal tissues. This investigation identified somatic truncating mutations affecting the prolactin receptor (PRLR) in all tumor and no normal samples. Targeted sequencing confirmed the presence of these mutations in precancerous lesions, indicating that this is an early event in tumorigenesis. Functional evaluation of these heterozygous mutations in Stat1(-/-) mouse embryonic fibroblasts showed that co-expression of truncated and wild-type PRLR led to aberrant STAT3 and STAT5 activation downstream of the receptor, cellular transformation in vitro, and tumor formation in vivo. In conclusion, truncating mutations of PRLR promote tumor growth in a model of human ERα+ breast cancer and warrant further investigation.
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http://dx.doi.org/10.1016/j.celrep.2016.08.076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557050PMC
September 2016

Comprehensive genomic analysis reveals FLT3 activation and a therapeutic strategy for a patient with relapsed adult B-lymphoblastic leukemia.

Exp Hematol 2016 Jul 13;44(7):603-13. Epub 2016 May 13.

Siteman Cancer Center, Washington University, St. Louis, MO, USA; Department of Medicine, Washington University, St. Louis, MO, USA.

The genomic events responsible for the pathogenesis of relapsed adult B-lymphoblastic leukemia (B-ALL) are not yet clear. We performed integrative analysis of whole-genome, whole-exome, custom capture, whole-transcriptome (RNA-seq), and locus-specific genomic assays across nine time points from a patient with primary de novo B-ALL. Comprehensive genome and transcriptome characterization revealed a dramatic tumor evolution during progression, yielding a tumor with complex clonal architecture at second relapse. We observed and validated point mutations in EP300 and NF1, a highly expressed EP300-ZNF384 gene fusion, a microdeletion in IKZF1, a focal deletion affecting SETD2, and large deletions affecting RB1, PAX5, NF1, and ETV6. Although the genome analysis revealed events of potential biological relevance, no clinically actionable treatment options were evident at the time of the second relapse. However, transcriptome analysis identified aberrant overexpression of the targetable protein kinase encoded by the FLT3 gene. Although the patient had refractory disease after salvage therapy for the second relapse, treatment with the FLT3 inhibitor sunitinib rapidly induced a near complete molecular response, permitting the patient to proceed to a matched-unrelated donor stem cell transplantation. The patient remains in complete remission more than 4 years later. Analysis of this patient's relapse genome revealed an unexpected, actionable therapeutic target that led to a specific therapy associated with a rapid clinical response. For some patients with relapsed or refractory cancers, this approach may indicate a novel therapeutic intervention that could alter outcome.
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http://dx.doi.org/10.1016/j.exphem.2016.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914477PMC
July 2016

Optimizing cancer genome sequencing and analysis.

Cell Syst 2015 Sep;1(3):210-223

The McDonnell Genome Institute, Washington University, St. Louis, MO, USA, 63108 ; Department of Genetics, Washington University, St. Louis, MO, USA, 63108 ; Siteman Cancer Center, Washington University, St. Louis, MO, USA, 63108 ; Department of Medicine, Washington University, St. Louis, MO, USA, 63108.

Tumors are typically sequenced to depths of 75-100× (exome) or 30-50× (whole genome). We demonstrate that current sequencing paradigms are inadequate for tumors that are impure, aneuploid or clonally heterogeneous. To reassess optimal sequencing strategies, we performed ultra-deep (up to ~312×) whole genome sequencing (WGS) and exome capture (up to ~433×) of a primary acute myeloid leukemia, its subsequent relapse, and a matched normal skin sample. We tested multiple alignment and variant calling algorithms and validated ~200,000 putative SNVs by sequencing them to depths of ~1,000×. Additional targeted sequencing provided over 10,000× coverage and ddPCR assays provided up to ~250,000× sampling of selected sites. We evaluated the effects of different library generation approaches, depth of sequencing, and analysis strategies on the ability to effectively characterize a complex tumor. This dataset, representing the most comprehensively sequenced tumor described to date, will serve as an invaluable community resource (dbGaP accession id phs000159).
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http://dx.doi.org/10.1016/j.cels.2015.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669575PMC
September 2015

DGIdb 2.0: mining clinically relevant drug-gene interactions.

Nucleic Acids Res 2016 Jan 3;44(D1):D1036-44. Epub 2015 Nov 3.

McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO 63110, USA Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA

The Drug-Gene Interaction Database (DGIdb, www.dgidb.org) is a web resource that consolidates disparate data sources describing drug-gene interactions and gene druggability. It provides an intuitive graphical user interface and a documented application programming interface (API) for querying these data. DGIdb was assembled through an extensive manual curation effort, reflecting the combined information of twenty-seven sources. For DGIdb 2.0, substantial updates have been made to increase content and improve its usefulness as a resource for mining clinically actionable drug targets. Specifically, nine new sources of drug-gene interactions have been added, including seven resources specifically focused on interactions linked to clinical trials. These additions have more than doubled the overall count of drug-gene interactions. The total number of druggable gene claims has also increased by 30%. Importantly, a majority of the unrestricted, publicly-accessible sources used in DGIdb are now automatically updated on a weekly basis, providing the most current information for these sources. Finally, a new web view and API have been developed to allow searching for interactions by drug identifiers to complement existing gene-based search functionality. With these updates, DGIdb represents a comprehensive and user friendly tool for mining the druggable genome for precision medicine hypothesis generation.
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http://dx.doi.org/10.1093/nar/gkv1165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702839PMC
January 2016

Reduced levels of Hspa9 attenuate Stat5 activation in mouse B cells.

Exp Hematol 2015 Apr 27;43(4):319-30.e10. Epub 2014 Dec 27.

Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA; Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA; Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA; Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO, USA. Electronic address:

HSPA9 is located on chromosome 5q31.2 in humans, a region that is commonly deleted in patients with myeloid malignancies [del(5q)], including myelodysplastic syndrome (MDS). HSPA9 expression is reduced by 50% in patients with del(5q)-associated MDS, consistent with haploinsufficient levels. Zebrafish mutants and knockdown studies in human and mouse cells have implicated a role for HSPA9 in hematopoiesis. To comprehensively evaluate the effects of Hspa9 haploinsufficiency on hematopoiesis, we generated an Hspa9 knockout mouse model. Although homozygous knockout of Hspa9 is embryonically lethal, mice with heterozygous deletion of Hspa9 (Hspa9(+/-)) are viable and have a 50% reduction in Hspa9 expression. Hspa9(+/-) mice have normal basal hematopoiesis and do not develop MDS. However, Hspa9(+/-) mice have a cell-intrinsic reduction in bone marrow colony-forming unit-PreB colony formation without alterations in the number of B-cell progenitors in vivo, consistent with a functional defect in Hspa9(+/-) B-cell progenitors. We further reduced Hspa9 expression (<50%) using RNA interference and observed reduced B-cell progenitors in vivo, indicating that appropriate levels (≥50%) of Hspa9 are required for normal B lymphopoiesis in vivo. Knockdown of Hspa9 in an interleukin 7 (IL-7)-dependent mouse B-cell line reduced signal transducer and activator of transcription 5 (Stat5) phosphorylation following IL-7 receptor stimulation, supporting a role for Hspa9 in Stat5 signaling in B cells. Collectively, these data imply a role for Hspa9 in B lymphopoiesis and Stat5 activation downstream of IL-7 signaling.
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http://dx.doi.org/10.1016/j.exphem.2014.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375022PMC
April 2015

Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes.

Nat Genet 2011 Dec 11;44(1):53-7. Epub 2011 Dec 11.

Department of Internal Medicine, Division of Oncology, Washington University, St. Louis, Missouri, USA.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.
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http://dx.doi.org/10.1038/ng.1031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3247063PMC
December 2011

Knockdown of Hspa9, a del(5q31.2) gene, results in a decrease in hematopoietic progenitors in mice.

Blood 2011 Feb 1;117(5):1530-9. Epub 2010 Dec 1.

Department of Medicine, Division of Oncology, Washington University School of Medicine, 660 South Euclid Ave., St Louis, MO 63110, USA.

Heterozygous deletions spanning chromosome 5q31.2 occur frequently in the myelodysplastic syndromes (MDS) and are highly associated with progression to acute myeloid leukemia (AML) when p53 is mutated. Mutagenesis screens in zebrafish and mice identified Hspa9 as a del(5q31.2) candidate gene that may contribute to MDS and AML pathogenesis, respectively. To test whether HSPA9 haploinsufficiency recapitulates the features of ineffective hematopoiesis observed in MDS, we knocked down the expression of HSPA9 in primary human hematopoietic cells and in a murine bone marrow-transplantation model using lentivirally mediated gene silencing. Knockdown of HSPA9 in human cells significantly delayed the maturation of erythroid precursors, but not myeloid or megakaryocytic precursors, and suppressed cell growth by 6-fold secondary to an increase in apoptosis and a decrease in the cycling of cells compared with control cells. Erythroid precursors, B lymphocytes, and the bone marrow progenitors c-kit(+)/lineage(-)/Sca-1(+) (KLS) and megakaryocyte/erythrocyte progenitor (MEP) were significantly reduced in a murine Hspa9-knockdown model. These abnormalities suggest that cooperating gene mutations are necessary for del(5q31.2) MDS cells to gain clonal dominance in the bone marrow. Our results demonstrate that Hspa9 haploinsufficiency alters the hematopoietic progenitor pool in mice and contributes to abnormal hematopoiesis.
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http://dx.doi.org/10.1182/blood-2010-06-293167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056590PMC
February 2011

Lipophilic pyridinium bisphosphonates: potent gammadelta T cell stimulators.

Angew Chem Int Ed Engl 2010 Feb;49(6):1136-8

Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.

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http://dx.doi.org/10.1002/anie.200905933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2819003PMC
February 2010

Lipophilic bisphosphonates as dual farnesyl/geranylgeranyl diphosphate synthase inhibitors: an X-ray and NMR investigation.

J Am Chem Soc 2009 Apr;131(14):5153-62

Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.

Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anticancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth and how cell activity can be predicted based on enzyme inhibition data, and using X-ray diffraction, solid state NMR, and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.
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http://dx.doi.org/10.1021/ja808285eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753403PMC
April 2009

Bisphosphonate inhibitors of ATP-mediated HIV-1 reverse transcriptase catalyzed excision of chain-terminating 3'-azido, 3'-deoxythymidine: a QSAR investigation.

Bioorg Med Chem 2008 Oct 27;16(19):8959-67. Epub 2008 Aug 27.

Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.

We report the results of an investigation of the inhibition of the ATP-mediated HIV-1 reverse transcriptase catalyzed phosphorolysis in vitro of AZT from AZT-terminated DNA primers by a series of 42 bisphosphonates. The four most active compounds possess neutral, halogen-substituted phenyl or biphenyl sidechains and have IC(50) values < 1 microM in excision inhibition assays. Use of two comparative molecular similarity analysis methods to analyze these inhibition results yielded a classification model with an overall accuracy of 94%, and a regression model having good accord with experiment (q(2)=0.63, r(2)=0.91), with the experimental activities being predicted within, on average, a factor of 2. The most active species had little or no toxicity against three human cell lines (IC(50)(avg) > 200 microM). These results are of general interest since they suggest that it may be possible to develop potent bisphosphonate-based AZT-excision inhibitors with little cellular toxicity, opening up a new route to restoring AZT sensitivity in otherwise resistant HIV-1 strains.
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http://dx.doi.org/10.1016/j.bmc.2008.08.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2586422PMC
October 2008

Activity of sulfonium bisphosphonates on tumor cell lines.

J Med Chem 2007 Nov 27;50(24):6067-79. Epub 2007 Oct 27.

Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.

We investigated three series of sulfonium bisphosphonates for their activity in inhibiting the growth of three human tumor cell lines. The first series consisted of 6 cyclic sulfonium bisphosphonates, the most active species having an (average) IC50 of 89 microM. The second consisted of 10 phenylalkyl and phenylalkoxy bisphosphonates, the most active species having an IC50 of 18 microM. The third series consisted of 17 n-alkyl sulfonium bisphosphonates, the most active species having an IC50 of approximately 240 nM. Three QSAR models showed that the experimental cell growth inhibition results could be well predicted. We also determined the structures of one sulfonium bisphosphonate bound to farnesyl diphosphate synthase, finding that it binds exclusively to the dimethylallyl diphosphate binding site. These results are of interest since they show that sulfonium bisphosphonates can have potent activity against a variety of tumor cell lines, the most active species having IC50 values much lower than conventional nitrogen-containing bisphosphonates.
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http://dx.doi.org/10.1021/jm700991kDOI Listing
November 2007