Noah A. Brown

High prevalence of somatic MAP2K1 mutations in BRAF V600E negative Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) represents a clonal proliferation of Langerhans cells. BRAF V600E mutations have been identified in approximately 50% of cases. To discover other genetic mechanisms underlying LCH pathogenesis, we studied 8 cases of LCH using a targeted next-generation sequencing platform. An E102_I103del mutation in MAP2K1 was identified in one BRAF wild-type case and confirmed by Sanger sequencing. Analysis of 32 additional cases using BRAF V600E allele-specific polymerase chain reaction and Sanger sequencing of MAP2K1 exons 2 and 3 revealed somatic, mutually exclusive BRAF and MAP2K1 mutations in 18 of 40 (45.0%) and 11 of 40 (27.5%) cases, respectively. This is the first report of MAP2K1 mutations in LCH that occur in 50% of BRAF wild-type cases. The mutually exclusive nature of MAP2K1 and BRAF mutations implicates a critical role of oncogenic MAPK signaling in LCH. This finding may also have implications in the use of BRAF and MEK inhibitor therapy.

Activating FGFR2–RAS–BRAF Mutations in Ameloblastoma

Purpose: Ameloblastoma is an odontogenic neoplasm whose overall mutational landscape has not been well characterized. We sought to characterize pathogenic mutations in ameloblastoma and their clinical and functional significance with an emphasis on the mitogen-activated protein kinase (MAPK) pathway. Experimental Design: A total of 84 ameloblastomas and 40 non-ameloblastoma odontogenic tumors were evaluated with a combination of BRAF V600E allele–specific PCR, VE1 immunohistochemistry, the Ion AmpliSeq Cancer Hotspot Panel, and Sanger sequencing. Efficacy of a BRAF inhibitor was evaluated in an ameloblastoma-derived cell line. Results: Somatic, activating, and mutually exclusive RAS–BRAF and FGFR2 mutations were identified in 88% of cases. Somatic mutations in SMO, CTNNB1, PIK3CA, and SMARCB1 were also identified. BRAF V600E was the most common mutation, found in 62% of ameloblastomas and in ameloblastic fibromas/fibrodentinomas but not in other odontogenic tumors. This mutation was associated with a younger age of onset, whereas BRAF wild-type cases arose more frequently in the maxilla and showed earlier recurrences. One hundred percent concordance was observed between VE1 immunohistochemistry and molecular detection of BRAF V600E mutations. Ameloblastoma cells demonstrated constitutive MAPK pathway activation in vitro. Proliferation and MAPK activation were potently inhibited by the BRAF inhibitor vemurafenib. Conclusions: Our findings suggest that activating FGFR2–RAS–BRAF mutations play a critical role in the pathogenesis of most cases of ameloblastoma. Somatic mutations in SMO, CTNNB1, PIK3CA, and SMARCB1 may function as secondary mutations. BRAF V600E mutations have both diagnostic and prognostic implications. In vitro response of ameloblastoma to a BRAF inhibitor suggests a potential role for targeted therapy. Clin Cancer Res; 20(21); 5517–26. ©2014 AACR.

A novel recurrent NPM1-TYK2 gene fusion in cutaneous CD30-positive lymphoproliferative disorders

The spectrum of cutaneous CD30-positive lymphoproliferative disorders (LPDs) includes lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma. Chromosomal translocations targeting tyrosine kinases in CD30-positive LPDs have not been described. Using whole-transcriptome sequencing, we identified a chimeric fusion involving NPM1 (5q35) and TYK2 (19p13) that encodes an NPM1-TYK2 protein containing the oligomerization domain of NPM1 and an intact catalytic domain in TYK2. Fluorescence in situ hybridization revealed NPM1-TYK2 fusions in 2 of 47 (4%) primary cases of CD30-positive LPDs and was absent in other mature T-cell neoplasms (n = 151). Functionally, NPM1-TYK2 induced constitutive TYK2, signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5 activation. Conversely, a kinase-defective NPM1-TYK2 mutant abrogated STAT1/3/5 signaling. Finally, short hairpin RNA-mediated silencing of TYK2 abrogated lymphoma cell growth. This is the first report of recurrent translocations involving TYK2, and it highlights the novel therapeutic opportunities in the treatment of CD30-positive LPDs with TYK2 translocations.

Langerhans Cell Histiocytosis: A Clinicopathologic Review and Molecular Pathogenetic Update.

Langerhans cell histiocytosis (LCH) comprises a wide spectrum of clinical disorders that have in common a proliferation of Langerhans-type cells with characteristic morphologic, immunophenotypic, and ultrastructural features. In part because of the diverse clinical manifestations of LCH, there has long been controversy over whether LCH is best considered a reactive process or a neoplasm. Herein, we discuss the clinical and pathologic features of LCH, including recent advances in the understanding of the molecular pathogenesis of this disease that support its categorization as a neoplasm. We also review the implications that these recently described molecular characteristics may have on risk stratification and treatment of LCH.

Ameloblastoma: A Review of Recent Molecular Pathogenetic Discoveries.

Ameloblastoma is an odontogenic neoplasm whose molecular pathogenesis has only recently been elucidated. The discovery of recurrent activating mutations in FGFR2, BRAF, and RAS in a large majority of ameloblastomas has implicated dysregulation of MAPK pathway signaling as a critical step in the pathogenesis of this tumor. Some degree of controversy exists regarding the role of mutations affecting the sonic hedgehog (SHH) pathway, specifically Smoothened (SMO), which have been postulated to serve as either an alternative pathogenetic mechanism or secondary mutations. Here, we review recent advances in our understanding of the molecular pathogenesis of ameloblastoma as well as the diagnostic, prognostic, and therapeutic implications of these discoveries.

Evaluation of Allele-Specific PCR and Immunohistochemistry for the Detection of BRAF V600E Mutations in Hairy Cell Leukemia

OBJECTIVES Detection of BRAF V600E mutations in hairy cell leukemia (HCL) has important diagnostic utility. In this study, we sought to compare immunohistochemistry with an antibody specific for this mutation to a sensitive molecular assay. METHODS The performance of the BRAF V600E-specific VE1 antibody was compared with that of allele-specific polymerase chain reaction (PCR) in 22 formalin-fixed, paraffin-embedded (FFPE) specimens with HCL involvement, along with nine splenic marginal zone lymphomas (SMZLs), 10 follicular lymphomas (FLs), 10 mantle cell lymphomas (MCLs), and 10 chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLLs). An additional 11 SMZLs, 100 FLs, 20 MCLs, 83 CLL/SLL specimens, and 49 reactive tonsils within tissue microarrays were stained with VE1. RESULTS A BRAF V600E mutation was detected in 17 (77.3%) of 22 HCL cases by PCR. Immunohistochemistry demonstrated VE1 staining in 20 (90.9%) cases, identifying low-level (~1%) involvement in three HCL cases that were mutation negative by PCR. Evaluation of additional material from these patients confirmed the presence of BRAF V600E. Thirty-nine non-HCL cases were negative by both methods. Within tissue microarrays, weak false-positive staining was observed in two (0.8%) of 263 non-HCL cases. CONCLUSIONS VE1 immunohistochemistry is more sensitive than allele-specific PCR in FFPE bone marrow specimens and can be applied to decalcified core biopsy specimens that are not appropriate for molecular techniques.

Diagnosis of Clostridium difficile infection: Comparison of four methods on specimens collected in Cary-Blair transport medium and tcdB PCR on fresh versus frozen samples

Clostridium difficile infection (CDI) caused by toxigenic strains of C. difficile is primarily a nosocomial infection with increasing prevalence. Stool specimens are typically collected in Cary-Blair transport medium to maximize culture-based detection of common stool pathogens. The goal of this study was to establish an analytically accurate and efficient algorithm for the detection of CDI in our patient population using samples collected in Cary-Blair transport medium. In addition, we wished to determine whether the sensitivity and specificity of PCR was affected by freezing samples before testing. Using 357 specimens, we compared four methods: enzyme immunoassay for the antigen glutamate dehydrogenase (Wampole™ C. DIFF CHEK-60 Assay, GDH), toxin A and B enzyme immunoassay (Remel ProSpecT™ C. difficile Toxin A/B Microplate Assay, Toxin EIA), cell culture cytotoxicity neutralization assay (Bartels™ Cytotoxicity Assay, CT), and real-time PCR targeting the toxin B gene (BD GeneOhm™ Cdiff Assay, PCR). The analytic sensitivity and specificity of each as determined using a combined gold standard were as follows: GDH, 100% and 93.2%; Toxin EIA, 82.9% and 82.9%; CT, 100% and 100%; PCR (performed on frozen specimens) 74.3% and 96.6%; respectively. However, the sensitivity and specificity of PCR improved to 100% when performed on 50 fresh stool samples collected in Cary-Blair. While CT remains a sensitive method for the detection of CDI, GDH offers an excellent initial screening method to rule out CDI. While the performance of each assay did not appear to be affected by collection in Cary-Blair medium, PCR performed better using fresh specimens.

Activating KRAS mutations are characteristic of oncocytic sinonasal papilloma and associated sinonasal squamous cell carcinoma.

Oncocytic sinonasal papillomas (OSPs) are benign tumours of the sinonasal tract, a subset of which are associated with synchronous or metachronous sinonasal squamous cell carcinoma (SNSCC). Activating EGFR mutations were recently identified in nearly 90% of inverted sinonasal papillomas (ISPs) – a related tumour with distinct morphology. EGFR mutations were, however, not found in OSP, suggesting that different molecular alterations drive the oncogenesis of these tumours. In this study, tissue from 51 cases of OSP and five cases of OSP‐associated SNSCC was obtained retrospectively from six institutions. Tissue was also obtained from 50 cases of ISP, 22 cases of ISP‐associated SNSCC, ten cases of exophytic sinonasal papilloma (ESP), and 19 cases of SNSCC with no known papilloma association. Using targeted next‐generation and conventional Sanger sequencing, we identified KRAS mutations in 51/51 (100%) OSPs and 5/5 (100%) OSP‐associated SNSCCs. The somatic nature of KRAS mutations was confirmed in a subset of cases with matched germline DNA, and four matched pairs of OSP and concurrent associated SNSCC had concordant KRAS genotypes. In contrast, KRAS mutations were present in only one (5%) SNSCC with no known papilloma association and none of the ISPs, ISP‐associated SNSCCs, or ESPs. This is the first report of somatic KRAS mutations in OSP and OSP‐associated SNSCC. The presence of identical mutations in OSP and concurrent associated SNSCC supports the putative role of OSP as a precursor to SNSCC, and the high frequency and specificity of KRAS mutations suggest that OSP and OSP‐associated SNSCC are biologically distinct from other similar sinonasal tumours. The identification of KRAS mutations in all studied OSP cases represents an important development in our understanding of the pathogenesis of this disease and may have implications for diagnosis and therapy. Copyright

Functional proteogenomics reveals biomarkers and therapeutic targets in lymphomas

Significance An important goal in precision oncology is the identification of biomarkers and therapeutic targets. We identified and annotated a compendium of N-glycoproteins from diverse human lymphoid neoplasia, an attractive class of proteins with potential to serve as cancer biomarkers and therapeutic targets. In anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL), integration of N-glycoproteomics and transcriptome sequencing revealed an underappreciated and targetable ALK-regulated cytokine/receptor signaling network highlighting the utility of functional proteogenomics for discovery of cancer biomarkers and therapeutic targets. Identification of biomarkers and therapeutic targets is a critical goal of precision medicine. N-glycoproteins are a particularly attractive class of proteins that constitute potential cancer biomarkers and therapeutic targets for small molecules, antibodies, and cellular therapies. Using mass spectrometry (MS), we generated a compendium of 1,091 N-glycoproteins (from 40 human primary lymphomas and cell lines). Hierarchical clustering revealed distinct subtype signatures that included several subtype-specific biomarkers. Orthogonal immunological studies in 671 primary lymphoma tissue biopsies and 32 lymphoma-derived cell lines corroborated MS data. In anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL), integration of N-glycoproteomics and transcriptome sequencing revealed an ALK-regulated cytokine/receptor signaling network, including vulnerabilities corroborated by a genome-wide clustered regularly interspaced short palindromic screen. Functional targeting of IL-31 receptor β, an ALCL-enriched and ALK-regulated N-glycoprotein in this network, abrogated ALK+ALCL growth in vitro and in vivo. Our results highlight the utility of functional proteogenomic approaches for discovery of cancer biomarkers and therapeutic targets.

Requisite analytic and diagnostic performance characteristics for the clinical detection of BRAF V600E in hairy cell leukemia: a comparison of 2 allele-specific PCR assays.

Detection of high-frequency BRAF V600E mutations in hairy cell leukemia (HCL) has important diagnostic utility. However, the requisite analytic performance for a clinical assay to routinely detect BRAF V600E mutations in HCL has not been clearly defined. In this study, we sought to determine the level of analytic sensitivity needed for formalin-fixed, paraffin-embedded (FFPE) and frozen samples and to compare the performance of 2 allele-specific polymerase chain reaction (PCR) assays. Twenty-nine cases of classic HCL, including 22 FFPE bone marrow aspirates and 7 frozen specimens from blood or bone marrow were evaluated using a laboratory-developed allele-specific PCR assay and a commercially available allele-specific quantitative PCR assay—myT BRAF Ultra. Also included were 6 HCL variant and 40 non-HCL B-cell lymphomas. Two cases of classic HCL, 1 showing CD5 expression, were truly BRAF V600E-negative based on negative results by PCR and sequencing despite high-level leukemic involvement. Among the remaining 27 specimens, V600E mutations were detected in 88.9% (17/20 FFPE; 7/7 frozen) and 81.5% (15/20 FFPE; 7/7 frozen), for the laboratory-developed and commercial assays, respectively. No mutations were detected among the 46 non-HCL lymphomas. Both assays showed an analytic sensitivity of 0.3% involvement in frozen specimens and 5% in FFPE tissue. On the basis of these results, an assay with high analytic sensitivity is required for the clinical detection of V600E mutations in HCL specimens. Two allele-specific PCR assays performed well in both frozen and FFPE bone marrow aspirates, although detection in FFPE tissue required 5% or more involvement.