Search

Supplementary Figure from Distinct Patterns of Clonal Evolution Drive Myelodysplastic Syndrome Progression to Secondary Acute Myeloid Leukemia

Supplementary Table from Distinct Patterns of Clonal Evolution Drive Myelodysplastic Syndrome Progression to Secondary Acute Myeloid Leukemia

Clonal evolution in myelodysplastic syndrome (MDS) can result in clinical progression and secondary acute myeloid leukemia (sAML). To dissect changes in clonal architecture associated with this progression, we performed single-cell genotyping of paired MDS and sAML samples from 18 patients. Analysis of single-cell genotypes revealed patient-specific clonal evolution and enabled the assessment of single-cell mutational cooccurrence. We discovered that changes in clonal architecture proceed via distinct patterns, classified as static or dynamic, with dynamic clonal architectures having a more proliferative phenotype by blast count fold change. Proteogenomic analysis of a subset of patients confirmed that pathogenic mutations were primarily confined to primitive and mature myeloid cells, though we also identify rare but present mutations in lymphocyte subsets. Single-cell transcriptomic analysis of paired sample sets further identified gene sets and signaling pathways involved in two cases of progression. Together, these data define serial changes in the MDS clonal landscape with clinical and therapeutic implications.Significance:Precise clonal trajectories in MDS progression are made possible by single-cell genomic sequencing. Here we use this technology to uncover the patterns of clonal architecture and clonal evolution that drive the transformation to secondary AML. We further define the phenotypic and transcriptional changes of disease progression at the single-cell level.See related article by Menssen et al., p. 330 (31).See related commentary by Romine and van Galen, p. 270.This article is highlighted in the In This Issue feature, p. 265

Large B cell lymphomas (LBCL) are a heterogeneous group of diseases with variable presentations and prognoses. Rarely, LBCLs arise in the sinonasal tract and are distinct from extranodal NK/T-cell lymphoma which is more typical in this anatomic location. We hypothesized that large B cell lymphoma primary to the sinonasal tract (snLBCL) would be heterogeneous and include high-grade B cell lymphomas (HGBCL) described in the revised 4th edition WHO classification of lymphomas. We retrospectively evaluated cases of snLBCL at our center, and performed additional immunohistochemical and in situ hybridization studies where needed for modern WHO classification. Our cohort consisted of 25 cases, 15 males and 10 female patients, aged 14 to 87 years, with predominantly nasopharyngeal disease (n = 11), Ann Arbor stage IIE (n = 15), and immunocompetence (n = 24). According to revised 2016 WHO criteria, 20 of the 25 cases were DLBCL-NOS (80%, two-thirds germinal center phenotype), 3 were HGBCL-NOS (8%, one with MYC rearrangement without BCL2 rearrangement), and 2 were EBV-LBCL (8%). Among DLBCL-NOS, those arising in the nasopharynx all showed a germinal center B cell (GCB) phenotype, whereas both evaluable maxillary sinus tumors showed non-GCB characteristics (p = 0.02). These data show that large B cell lymphoma primary to the sinonasal tract is histopathologically heterogeneous as systemic large B cell lymphoma. The observation that GCB and non-GCB tumors differs in anatomic location suggests that microenvironmental factors in sinonasal anatomic sites may drive lymphoma characteristics.

Clonal evolution in myelodysplastic syndrome (MDS) can result in clinical progression and secondary acute myeloid leukemia (sAML). To dissect changes in clonal architecture associated with this progression, we performed single-cell genotyping of paired MDS and sAML samples from 18 patients. Analysis of single-cell genotypes revealed patient-specific clonal evolution and enabled the assessment of single-cell mutational cooccurrence. We discovered that changes in clonal architecture proceed via distinct patterns, classified as static or dynamic, with dynamic clonal architectures having a more proliferative phenotype by blast count fold change. Proteogenomic analysis of a subset of patients confirmed that pathogenic mutations were primarily confined to primitive and mature myeloid cells, though we also identify rare but present mutations in lymphocyte subsets. Single-cell transcriptomic analysis of paired sample sets further identified gene sets and signaling pathways involved in two cases of progression. Together, these data define serial changes in the MDS clonal landscape with clinical and therapeutic implications.Significance:Precise clonal trajectories in MDS progression are made possible by single-cell genomic sequencing. Here we use this technology to uncover the patterns of clonal architecture and clonal evolution that drive the transformation to secondary AML. We further define the phenotypic and transcriptional changes of disease progression at the single-cell level.See related article by Menssen et al., p. 330 (31).See related commentary by Romine and van Galen, p. 270.This article is highlighted in the In This Issue feature, p. 265

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder that causes a predisposition to develop tumors along the peripheral nervous system. The NF1 gene, located at 17q11.2, has the highest mutation rate among known human genes and about half of NF1 patients have de novo pathogenic variants. We present a case of clinical NF1 diagnoses in multiple family members with phenotypes ranging from mild to severe. Chromosome analysis of the 3-year-old female proband with NF1 resulted in an abnormal karyotype that was inherited from her mother: 46,XX,t(4;17)(q21.3;q11.2) mat. However, no NF1 genetic variants were identified by either NGS analysis of NF1 DNA coding regions, deletion-duplication studies, or by cytogenomic microarray copy number analysis. Follow-up chromosome studies of the proband's two male siblings demonstrated cosegregation of the same balanced translocation and a clinical diagnosis of NF1. Based on the cosegregation of the translocation with the NF1 clinical presentation in this family, we hypothesized that the NF1 gene may have been disrupted by this unique rearrangement. Subsequent fluorescence in situ hybridization (FISH) analysis of the metaphase cells of an affected sibling revealed a disruption of the NF1 gene confirming the underlying basis of the clinical NF1 presentation in this family. The utilization of traditional cytogenetic as well as evolving molecular methods was not only pivotal in the diagnosis of NF1 and management for this family, but is also pertinent to other patients with a family history of NF1.

Multiple studies have demonstrated the utility of chromosomal microarray (CMA) testing to identify clinically significant copy number alterations (CNAs) and copy-neutral loss-of-heterozygosity (CN-LOH) in myeloid malignancies. However, guidelines for integrating CMA as a standard practice for diagnostic evaluation, assessment of prognosis and predicting treatment response are still lacking. CMA has not been recommended for clinical work-up of myeloid malignancies by the WHO 2016 or the NCCN 2017 guidelines but is a suggested test by the European LeukaemiaNet 2013 for the diagnosis of primary myelodysplastic syndrome (MDS). The Cancer Genomics Consortium (CGC) Working Group for Myeloid Neoplasms systematically reviewed peer-reviewed literature to determine the power of CMA in (1) improving diagnostic yield, (2) refining risk stratification, and (3) providing additional genomic information to guide therapy. In this manuscript, we summarize the evidence base for the clinical utility of array testing in the workup of MDS, myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and myeloproliferative neoplasms (MPN). This review provides a list of recurrent CNAs and CN-LOH noted in this disease spectrum and describes the clinical significance of the aberrations and how they complement gene mutation findings by sequencing. Furthermore, for new or suspected diagnosis of MDS or MPN, we present suggestions for integrating genomic testing methods (CMA and mutation testing by next generation sequencing) into the current standard-of-care clinical laboratory testing (karyotype, FISH, morphology, and flow).

Background Plasma cell neoplasm and/or plasma cell myeloma (PCM) is a mature B-cell lymphoproliferative neoplasm of plasma cells that secrete a single homogeneous immunoglobulin called paraprotein or M-protein. Plasma cells accumulate in the bone marrow (BM) leading to bone destruction and BM failure. Diagnosis of PCM is based on clinical, radiologic, and pathological characteristics. The percent of plasma cells by manual differential (bone marrow morphology), the white blood cell (WBC) count, cytogenetics, fluorescence in situ hybridization (FISH), microarray, and next-generation sequencing of BM are used in the risk stratification of newly diagnosed PCM patients. The genetics of PCM is highly complex and heterogeneous with several genetic subtypes that have different clinical outcomes. National Comprehensive Cancer Network guidelines recommend targeted FISH analysis of plasma cells with specific DNA probes to detect genetic abnormalities for the staging of PCM (4.2021). Recognition of risk categories through training software for classification of high-risk PCM and a novel way of addressing the current approaches through bioinformatics will be a significant step toward automation of PCM analysis. Methods A new artificial neural network (ANN) classification model was developed and tested in Python programming language with a first data set of 301 cases and a second data set of 176 cases for a total of 477 cases of PCM at diagnosis. Classification model was also developed with support vector machines (SVM) algorithm in R studio and interactive data visuals using Tableau. Results The resulting ANN algorithm had 94% accuracy for the first and second data sets with a classification summary of precision (PPV): 0.97, recall (sensitivity): 0.76, f1 score: 0.83, and accuracy of logistic regression of 1.0. SVM of plasma cells versus TP53 revealed a 95% accuracy level. Conclusion A novel classification model based only on specific morphological and genetic variables was developed using a machine learning algorithm, the ANN. ANN identified an association of WBC and BM plasma cell percentage with two of the high-risk genetic categories in the diagnostic cases of PCM. With further training and testing of additional data sets that include morphologic and additional genetic rearrangements, the newly developed ANN model has the potential to develop an accurate classification of high-risk categories of PCM.

Clinical management and risk stratification of B-lymphoblastic leukemia/ lymphoma (B-ALL/LBL) depend largely on identification of chromosomal abnormalities obtained using conventional cytogenetics and Fluorescence In Situ Hybridization (FISH) testing. In the last few decades, testing algorithms have been implemented to support an optimal risk-oriented therapy, leading to a large improvement in overall survival. In addition, large scale genomic studies have identified multiple aberrations of prognostic significance that are not routinely tested by existing modalities. However, as chromosomal microarray analysis (CMA) and next-generation sequencing (NGS) technologies are increasingly used in clinical management of hematologic malignancies, these abnormalities may be more readily detected. In this article, we have compiled a comprehensive, evidence-based review of the current B-ALL literature, focusing on known and published subtypes described to date. More specifically, we describe the role of various testing modalities in the diagnosis, prognosis, and therapeutic relevance. In addition, we propose a testing algorithm aimed at assisting laboratories in the most effective detection of the underlying genomic abnormalities.

Primary central nervous system lymphoma (PCNSL) patients have a poorer prognosis than systemic lymphoma. Gain-of-function MYD88 c.794T>C (p. L265P) mutation and programmed cell death-1 (PD-1) pathway alterations are potential targetable pathways. Our study objective was to determine the clinicopathologic correlates of MYD88 mutation and PD-1 alterations in PCNSL and the impact of Epstein-Barr virus (EBV) infection. We studied 53 cases including 13 EBV-associated (EBV(pos)) PCNSL, 49% harbored MYD88 mutation, none seen in EBV(pos) PCNSL. MYD88 protein expression did not correlate with MYD88 mutation. T-cell and macrophage infiltration was common. All PD-L1 positive tumors were EBV(pos). Two PD-L1 positive tumors showed 9p24.1/PD-L1 locus alterations by Fluorescence In Situ Hybridization. T cells and macrophages expressed PD-1 and/or PD-L1 in 98% and 83% cases, respectively. MYD88 mutation or protein expression and PD-1 or PD-L1 expression did not predict outcome. We hypothesize that EBV(pos) PCNSL has a distinct activation mechanism, independent of genetic alterations.

Objectives Acute myeloid leukemia (AML) is classified in part by recurrent cytogenetic abnormalities, often detected by both fluorescent in situ hybridization (FISH) and karyotype. The goal of this study was to assess the utility of FISH and karyotyping at diagnosis and follow-up. Methods Adult AML samples at diagnosis or follow-up with karyotype and FISH were identified. Concordance was determined, and clinical characteristics and outcomes for discordant results were evaluated. Results Karyotype and FISH results were concordant in 193 (95.0%) of 203 diagnostic samples. In 10 cases, FISH detected an abnormality, but karyotype was normal. Of these, one had a FISH result with clinical significance. In follow-up cases, 17 (8.1%) of 211 showed FISH-positive discordant results; most were consistent with low-level residual disease. Conclusions Clinically significant discordance between karyotype and AML FISH is uncommon. Consequently, FISH testing can safely be omitted from most of these samples. Focused FISH testing is more useful at follow-up, for minimal residual disease detection.

Background: Current evidence indicates that patients with Human Epidermal Growth Factor Receptor 2 (HER2)-positive Breast Cancer (BC) benefit from HER2-targeted therapies. Accurate determination of HER2 status is critical to ensure that the correct patients are offered targeted treatment while patients with HER2-negative tumors–who are unlikely to benefit from anti-HER2 therapy– are spared from the adverse effects of these costly drugs. Guidelines for performance and reporting of HER2 testing, first published in 2007 by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP), were updated in October 2013. The new reporting criteria are based on a combination of HER2:CEP17 ratio and average HER2 copy number. Methods: We retrospectively reviewed HER2 dual probe FISH test results sequentially performed by the Cytogenetics Laboratory at Vanderbilt University from January to May 2014 since implementation of the updated guidelines. The cases were then rescored using the 2007 guidelines. The clinicopathologic features of cases with a change in Her2 status after scoring with the 2013 guidelines were examined for statistical significance. Results: If the 266 performed HER2 FISH tests had been scored using the 2007 guidelines the results would have been the following: amplified (n = 44, 16%), equivocal (n = 28, 10%) and not amplified (n = 194, 70%). However, using the 2013 guidelines the cases were actually reported as follows: amplified (n=57, 21%), equivocal (n=22, 8.3%) and not amplified (n=187, 68%). Additionally, 18 cases demonstrated genetic heterogeneity in >25% of cells. The updated guidelines resulted in a change in Her2 status in 12% of tests (32/266; p less than 0.0001); 13 changed from equivocal to amplified, 13 cases changed from not amplified to equivocal and 6 cases changed from equivocal to not amplified. Cases with a change in Her2 status following implementation of the new guidelines were more likely to demonstrate genetic heterogeneity (28% [9/32] vs. 4% [9/234]; p less than 0.0001). Furthermore, hormone receptor (HR)-negative tumors scored as not amplified by the 2007 guidelines were more likely to undergo an upgrade in HER2 status under the 2013 guidelines than HR-positive tumors (26% [11/41] vs. 7% [11/153], p less than 0.0001). Conclusions: The 2013 reporting criteria, based on a combination of HER2:CEP17 ratio and average HER2 copy number, increase the number of patients eligible for HER2-targeted therapies while decreasing equivocal results. Tumors that demonstrate genetic heterogeneity for Her2 or are HR-negative account for a significant percentage of these newly eligible cases. Correlation with clinical response is required to confirm the proposed improved analytical validity of the updated guidelines. Clinical trials to evaluate the benefit of anti-Her2 therapy in patients with genetic heterogeneity are in the planning phase. Citation Format: Monica V Estrada, Jena M Giltnane, Ferrin C Wheeler, Ashwini Yenamandra, Vandana Abramson, Ingrid A Mayer, Julie Means, Brent Rexer, Ingrid M Meszoely, Melinda E Sanders. Genetic heterogeneity for Her2 accounts for a significant percentage of breast cancers changing Her2 status following implementation of the 2013 CAP/ASCO HER2 reporting guidelines [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-32.

The goal of this research paper is to propose an approach to uncovering important and previously unknown indicators that could prevent suicide by analyzing data released by the Centers for Disease Control and Prevention (CDC). The CDC data includes 75 variables that identify characteristics and demographics of the deceased. This paper describes the process of exploring data science methods to build a predictive model that could lead to prevention of suicide rate in USA. The data was explored with the Python programming language, R scripting language, RStudio and in the Tableau environment. Preliminary analysis of the CDC 2013 deaths by suicide data indicate that education was one of the important factors that lead to suicide; further analysis of this and other data may provide a new set of objective risk factors from one or more of the variables in this data set.

Acute erythroblastic leukemia AEL a rare subset of de novo AML with a complex rearrangement involving ETV locus and loss of RB locus

Sepsis affects millions of people worldwide each year. It occurs when a normal human immune response to a bacterial, viral or fungal infection becomes dysfunctional and triggers widespread inflammation that results in severe tissue damage that leads to organ failure, shock, and death. Sepsis, requires immediate treatment and has a high readmission rate for survivors. It is also one of the most expensive conditions to treat. In 2013, there were more than 1.6 million cases of sepsis in the United States with a financial cost of more than $23 billion. Sepsis was first described in antiquity, and given its current name, by the ancient Greek physician Hippocrates. Despite its long medical history, severity, and financial burden, the causes of sepsis are not well understood, and there is no standard approach to diagnosis and treatment. The definition of sepsis, the characterization of its clinical stages, and sepsis monitoring tools have changed three times in the past 25 years, most recently in March 2016. The universal adoption of this latest definition, sepsis-3, and a screening tool, qSOFA, are currently under debate in the medical community. A means to rapidly identify and treat sepsis could reduce the five million deaths due to sepsis each year worldwide. This paper reviews the evolution of the definition of sepsis and the controversy surrounding the sepsis-3 definition and the sepsis screening tool, qSOFA., Comment: 16 pages, 6 figures

Gene fusions involving the catalytic domain of tyrosine kinases (TKs) are found in a variety of hematological and solid tumor malignancies. Clinically, TK fusions have emerged as prime targets for therapy with small molecule kinase inhibitors. Unfortunately, identification of TK fusions has been hampered by experimental limitations. Here, we developed version 2.0 of a genomically based systematic kinase fusion screen and used it to detect a novel imatinib-sensitive C6orf204-PDGFRB fusion in a patient with precursor T lymphoblastic lymphoma (T-ALL) and an associated myeloproliferative neoplasm with eosinophilia. These data validate the ability of this targeted capture-sequencing approach to detect TK fusion events in small amounts of DNA extracted directly from patient samples.

SummaryHdac3 is essential for efficient DNA replication and DNA damage control. Deletion of Hdac3 impaired DNA repair and greatly reduced chromatin compaction and heterochromatin content. These defects corresponded to increases in histone H3K9,K14ac; H4K5ac; and H4K12ac in late S phase of the cell cycle, and histone deposition marks were retained in quiescent Hdac3-null cells. Liver-specific deletion of Hdac3 culminated in hepatocellular carcinoma. Whereas HDAC3 expression was downregulated in only a small number of human liver cancers, the mRNA levels of the HDAC3 cofactor NCOR1 were reduced in one-third of these cases. siRNA targeting of NCOR1 and SMRT (NCOR2) increased H4K5ac and caused DNA damage, indicating that the HDAC3/NCOR/SMRT axis is critical for maintaining chromatin structure and genomic stability.

DDB1, a component of a Cul4A ubiquitin ligase complex, promotes nucleotide excision repair (NER) and regulates DNA replication. We have investigated the role of human DDB1 in maintaining genome stability. DDB1-depleted cells accumulate DNA double-strand breaks in widely dispersed regions throughout the genome and have activated ATM and ATR cell cycle checkpoints. Depletion of Cul4A yields similar phenotypes, indicating that an E3 ligase function of DDB1 is important for genome maintenance. In contrast, depletion of DDB2, XPA, or XPC does not cause activation of DNA damage checkpoints, indicating that defects in NER are not involved. One substrate of DDB1-Cul4A that is crucial for preventing genome instability is Cdt1. DDB1-depleted cells exhibit increased levels of Cdt1 protein and rereplication, despite containing other Cdt1 regulatory mechanisms. The rereplication, accumulation of DNA damage, and activation of checkpoint responses in DDB1-depleted cells require entry into S phase and are partially, but not completely, suppressed by codepletion of Cdt1. Therefore, DDB1 prevents DNA lesions from accumulating in replicating human cells, in part by regulating Cdt1 degradation.

The 2008 WHO classification scheme of hematolymphoid neoplasms recognizes a category of myeloid and lymphoid neoplasms (MLNs) with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1.1 The postulated cell of origin for PDGFRA or FGFR1-rearranged diseases is a pluripotent progenitor capable of giving rise to myeloid neoplasms (myeloproliferative neoplasms and acute leukemias) and to lymphoblastic leukemia/lymphoma. Historically, PDGFRB translocations have not been associated with malignancies of the lymphoid lineage. Myeloid neoplasms with abnormalities of PDGFRB have been described to have hematologic features of chronic myelomonocytic leukemia (CMML), sometimes with eosinophilia, or as various other myeloproliferative and/or myelodysplastic neoplasms.2 In recognition of this distinction, the classification scheme designates specific categories of “MLNs” with PDGFRA and FGFR1 rearrangement, but omits the word “lymphoid” from the PDGFRB-associated entity.1 To call attention to the rare occurrence of lymphoid and mixed MLNs with abnormalities of PDGFRB, we present the clinical and pathological details of 2 cases.

Epstein Barr virus (EBV)-related hemophagocytic lymphohistiocytosis (EBV-HLH) is a form of acquired, infection-related HLH which typically represents a fulminant presentation of an acute EBV infection of CD8+ T cells with 30-50% mortality rate. Systemic EBV-positive lymphoproliferative disease of childhood (SE-LPD) is a rare T cell lymphoproliferative disorder predominantly arising in the setting of acute EBV infection, often presenting with HLH. Since both entities have been associated with clonal T cell populations, the discrimination between these diseases is often ambiguous. We report a unique case of a 21 years old female who presented with clinical and laboratory findings of florid HLH in the setting of markedly elevated EBV titers (>1 million) and an aberrant T cell population shown to be clonal by flow cytometry, karyotype, and molecular studies. This case raises the differential of EBV-HLH versus SE-LPD. Review of the literature identified 74 cases of reported EBV-HLH and 21 cases of SE-LPD with associated HLH in 25 studies. Of those cases with available outcome data, 62 of 92 cases (67%) were fatal. Of 60 cases in which molecular clonality was demonstrated, 37 (62%) were fatal, while all 14 cases (100%) demonstrating karyotypic abnormalities were fatal. Given the karyotypic findings in this sentinel case, a diagnosis of SE-LPD was rendered. The overlapping clinical and pathologic findings suggest that EBV-HLH and SE-LPD are a biologic continuum, rather than discrete entities. The most clinically useful marker of mortality was an abnormal karyotype rather than other standards of clonality assessment.

Breast cancer proliferation measured by Ki67 immunohistochemistry after short-term antiestrogen therapy has been shown to correlate with disease-free survival. This suggests the use of biomarkers of the early effects of endocrine therapy on ER+ tumors will identify resistant cancers. Thus, we hypothesized that profiling operable ER+ tumors after short term treatment with an aromatase inhibitor would discover actionable molecular alterations causally associated with resistance to estrogen deprivation. We performed whole exome sequencing, RNA-Seq and quantitative immunofluorescence (QIF) of ER, PR, HER2, and Ki67 in biopsies from 130 patients with an operable ER+/HER2– breast cancer that had received letrozole for 10-21 days prior to surgery. Tumors were categorized by the natural log of 2-week post-letrozole Ki67 as sensitive, intermediate, or resistant. We sequenced RNA from 50 frozen tumors and performed fusion transcript analysis using 4 programmatic algorithms (dRanger, TopHat, DeFuse, Chimera Scan), resulting in 304 candidate gene fusions in 44 tumors. Primers with universal sequencing tags were designed against 3’ and 5’ sites of breakpoints mapping to RefSeq exon coding regions (n=187); fusion sequences were amplified by qRT-PCR from tumor and breast cancer cell line RNA. Single or multiple distinct product bands were visualized by gel electrophoresis in 96 tumor samples and Sanger-sequenced. Results were mapped to the human RNA reference transcriptome using BLAST. Overall, 9% of putative fusion transcripts (n=27 from 16 unique tumors) were validated by mapping to the open reading frames of predicted 3’ and 5’ genes. Fusion transcripts called by more than one program were more likely to validate (13 of 24 redundant versus 14 of 269 unique; p Using the 2-week Ki67 to stratify for response to treatment, the validated ESR1 fusions were present only in tumors that maintained high (≥7.4%) to intermediate (>2.7%) Ki67 labeling indices upon estrogen deprivation with letrozole (p=0.01). PR expression was lower (p=0.003) and ER expression higher (p=0.05) in ESR1 fusion+ tumors compared to fusion negative tumors. RNA extracted from 14 additional tumors were screened for ESR1 fusions by qRT-PCR and the ESR1:CCDC170 fusion was validated in 1 of 8 resistant/intermediate and 0 of 6 sensitive tumors. In summary, biomarkers of early response to antiestrogens are needed in order to identify ER+ cancers that are treatment resistant. In a prospective trial of operable ER+/HER2− breast tumors, we discovered recurrent intrachromosomal ESR1 fusion transcripts associated with intrinsic resistance to estrogen deprivation with letrozole. Additional work investigating the genomic basis and function of the fusion transcripts is underway. Citation Format: Jennifer M Giltnane, Justin M Balko, Thomas L Stricker, Christian Young, M Valeria Estrada, Nikhil Wagle, Eliezer van Allen, X Jasmine Mu, Violeta Sanchez, Jaime Farley, Kerry Fitzgerald, Armin Graber, Joseph A Pinto, Franco Doimi, Henry Gómez, Monica Rizzo, Thomas B Julian, Vandana Abramson, Ingrid Mayer, Mark Kelley, Ashwini Yenamandra, Ferrin C Wheeler, Melinda Sanders, Levi Garraway, Ingrid Meszoely, Carlos L Arteaga. Recurrent ESR1 fusion transcripts are associated with endocrine resistance in estrogen receptor positive, HER2 negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-3.

Tyrosine kinase (TK) fusion proteins result from genomic rearrangements that juxtapose TK signaling domains with various upstream partners. Since tumor cells can become dependent upon the resultant constitutive kinase activity for tumorigenesis and survival, TK fusions serve as attractive therapeutic targets. However, identification of TK fusions in cancers has been hindered by experimental techniques that lack sufficient sensitivity and throughput. To overcome these challenges, we developed a screen to systematically identify TK fusions in tumor DNA. This method involves selective capture of regions upstream of GXGXXG kinase motifs where TK fusion breakpoints are likely to occur followed by next generation sequencing and custom computational analysis. This approach was previously validated using cell line DNA (Chmielecki et al, ‘10). Here, we used an improved version of our platform to identify a novel TK fusion using DNA isolated from whole blood of a patient with recurrent precursor T lymphoblastic lymphoma (T-ALL) who presented with new leukocytosis and bone marrow findings compatible with a myeloproliferative neoplasm (MPN) with eosinophilia. Cytogenetic analysis of a bone marrow aspirate prior to treatment for the patient's MPN revealed a karyotype with a t(5;6)(q33-34;q23). At recurrence, a 5q33/PDGFRβ rearrangement with a breakpoint proximal to the MYB locus on chromosome 6 was identified with fluorescence in situ hybridization (FISH). Prior to allogeneic hematopoietic cell transplantation, a brief trial of imatinib confirmed responsiveness of eosinophilia. To identify the exact fusion breakpoint, 3 µg of whole blood genomic DNA was subjected to genomic capture followed by paired-end SOLiD sequencing. Fusion candidates were validated by direct sequencing of PCR products generated with breakpoint spanning primers. Analysis of the recovered sequences identified a fusion involving c6orf204 upstream of the PDGFRβ TK domain. 5’-RACE confirmed expression of c6orf204-PDGFRβ mRNA in the patient's blood cells. Motif analysis of the 5’ c6orf204 sequence present within the fusion revealed a coiled-coil domain, which likely facilitates dimerization of the kinase, as observed in other TK fusions (e.g. BCR-ABL). Interestingly, retrospective FISH studies of the patient's diagnostic lymph node involved by T-ALL also revealed the presence of a PDGFRβ rearrangement. These data suggest that the rearrangement occurred in a multi-potent progenitor cell that gave rise to both the T-ALL and MPN. This finding is analogous to 8p MPNs involving FGFR1, but has not yet been reported with PDGFRβ. Further biochemical analysis of this TK fusion is currently underway, and additional tumor samples are undergoing screening for other novel TK fusions. This work was supported by an SU2C-AACR Innovative Research Grant to WP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4977. doi:10.1158/1538-7445.AM2011-4977