Katherine Bell

Abstract 2408: Identification of R-Spondin fusions in various types of human cancer

Autocrine or paracrine constitutive Wnt pathway activation occurs at a high frequency in several tumor types. The R-spondin (RSPO) protein family is comprised of four secreted growth factors. The four paralogs share 40-60% pairwise amino acid sequence identity and are predicted to share substantial structural homology. RSPO proteins are involved in vertebrate development and their ligand-type activities overlap substantially with those of canonical Wnt ligands. A characteristic feature of all four RSPO members is their ability to activate β-catenin signaling and enhance WNT-mediated β-catenin activation. It has recently been described that recurrent gene fusions involving RSPO family members RSPO2 and RSPO3 occur in ∼10% of colon tumors. In this study we developed a TaqMan qRT-PCR-based approach to evaluate the expression of these three (3) RSPO fusion transcripts in formalin-fixed paraffin embedded tissue (FFPET) samples. We examined 324 lung cancer, 81 colorectal cancer, 71 head & neck, 11 esophageal, 92 ovarian cancer, and 103 breast cancer FFPET samples for the presence of EIF3E(e1)-RSPO2(e1), PTPRK(e1)-RSPO3(e2), and PTPRK(e7)-RSPO3(e2). EIF3E(e1)-RSPO2(e1), a fusion which is expected to produce a functional RSPO2 protein driven by the EIF3E promoter, was identified in ∼1-2% of most of cancer types with the exception of breast cancer. The PTPRK(e1)-RSPO3(e2) fusion was expressed by ∼1-11% of the samples in the different cancers, making it the most prevalent of the fusions. PTPRK(e1)-RSPO3(e2) fusion is an in-frame fusion that preserves the entire coding sequence of RSPO3 and replaces its secretion signal sequence with that of PTPRK. The PTPRK(e7)-RSPO3(e2) fusion is also an in-frame fusion in which the RSPO3 native signal peptide is replaced by the secretion signal of PTPRK. The PTPRK(e7)-RSPO3(e2) was the least prevalent of all the fusions, positive samples were found exclusively in the head and neck (∼2%) and breast cancer samples (∼2%). All of the fusions detected were mutually exclusive. The RSPO gene fusions identified may provide new potential opportunities for therapeutic intervention. Citation Format: Gabriela Martinez Cardona, Katherine Bell, Joseph Portale, Dana Gaffney, Christopher Moy, Suso Platero, Matthew V. Lorenzi, Jayaprakash Karkera. Identification of R-Spondin fusions in various types of human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2408. doi:10.1158/1538-7445.AM2014-2408

Oncogenic Characterization and Pharmacologic Sensitivity of Activating Fibroblast Growth Factor Receptor (FGFR) Genetic Alterations to the Selective FGFR Inhibitor Erdafitinib

Fibroblast growth factor receptor (FGFR) genetic alterations are frequently observed in cancer, suggesting that FGFR inhibition may be a promising therapy in patients harboring these lesions. Identification of predictive and pharmacodynamic biomarkers to select and monitor patients most likely to respond to FGFR inhibition will be the key to clinical development of this class of agents. Sensitivity to FGFR inhibition and correlation with FGFR pathway activation status were determined in molecularly annotated panels of cancer cell lines and xenograft models. Pathway inhibition in response to FGFR inhibitor treatment was assessed in cell lines (both in vitro and in vivo) and in samples from patients treated with the FGFR inhibitor JNJ-42756493 (erdafitinib). Frequency of FGFR aberrations was assessed in a panel of NSCLC, breast, prostate, ovarian, colorectal, and melanoma human tumor tissue samples. FGFR translocations and gene amplifications present in clinical specimens were shown to display potent transforming activity associated with constitutive pathway activation. Tumor cells expressing these FGFR activating mutants displayed sensitivity to the selective FGFR inhibitor erdafitinib and resulted in suppression of FGFR phosphorylation and downstream signal transduction. Clinically, patients receiving erdafitinib showed decreased Erk phosphorylation in tumor biopsies and elevation of serum phosphate. In a phase I study, a heavily pretreated bladder cancer patient with an FGFR3–TACC3 translocation experienced a partial response when treated with erdafitinib. This preclinical study confirmed pharmacodynamics and identified new predictive biomarkers to FGFR inhibition with erdafitinib and supports further clinical evaluation of this compound in patients with FGFR genetic alterations. Mol Cancer Ther; 16(8); 1717–26. ©2017 AACR.

Abstract 5: Identification of androgen receptor (AR) splice variants, AR somatic mutations and TMPRSS2:ETS fusion genes in prostate cancer FFPET by qRT-PCR.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate Cancer (PCa) is the third most common cause of death from cancer in men of all ages. Surgical or medical castration is one of the most common treatments for patients with advanced PCa; however a majority of patients develop castration resistant prostate cancer (CRPC), tumor relapse, which remains to be the second leading cause of cancer-related deaths of men in the US. Androgen receptor (AR) signaling is shown to play a critical role in the development and progression of PCa. Genetic aberrations within AR, including constitutively active AR splice variants and AR point mutations have been identified in CRPC. The most common AR splice variants lack the ligand-binding domain (LBD), which is often the target of CRPC therapies. Therefore, presence of these variants may act as a mechanism of resistance to AR-targeted therapies leading to the progression of prostate tumor growth. Additional PCa specific genetic aberrations include fusions between the androgen-related gene, TMPRSS2 and the ETS transcription factors, ERG (predominant) and ETV1. These fusion events are frequently associated with more aggressive prostate cancers leading to poorer prognosis. In this study, we developed TaqMan qRT-PCR assays to evaluate the presence of several previously identified AR splice variants, including ARV1, ARV3/V7, ARV567 and ARV8, AR somatic mutations, including L701H, V715M, H874Y and T877A, along with TMPRSS2 fusion genes, TMPRSS2:ERG and TMPRSS2:ETV1, in two independent PCa FFPET sample sets. The first sample set consisted of 42 Prostate adenocarcinomas ranging from stage II to stage IV. Results showed that ARV1 and ARV3/V7 were the most prevalent variants with 92% of all samples showing expression of either or both variant. TMPRSS2: ERG was present in 72% of all samples tested, with a high concordance to AR variant expression, prevalent in later stage (III/IV) PCa samples. The second sample set consisted of 8 prostate adenocarcinomas, including matched adjacent normal FFPET. Similar expression of the AR variants was observed in both the tumor and matched normal samples, however tumor prostate samples showed a higher and more prevalent expression (66.67%) of the TMPRSS2: ERG fusion gene than in the matched normal samples (33%). None of the four AR mutations evaluated were detected in either sample set. Overall, these findings demonstrate a strong presence of both AR splice variants and the TMPRSS2: ERG fusion gene in the prostate cancer patient population, supporting evidence for a functional role of these markers in PCa diagnosis and disease progression. Furthermore, presence of LBD negative AR splice variants indicates an attractive biomarker for stratification of the patient population resistant to AR targeted therapies. Citation Format: Dana S. Gaffney, Gabriela Martinez, Katherine Bell, Suso Platero, Deborah Ricci, Jayaprakash Karkera. Identification of androgen receptor (AR) splice variants, AR somatic mutations and TMPRSS2:ETS fusion genes in prostate cancer FFPET by qRT-PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5. doi:10.1158/1538-7445.AM2013-5

Abstract 5027: Detection of soluble protein biomarkers in NSCLC serum samples by meso scale discovery electrochemiluminescence platform

Identification of soluble biomarkers has become a critical non-invasive approach for disease diagnosis and monitoring in the treatment of solid tumors. Non-Small Cell Lung Cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 85% of all lung cancers worldwide. The EGFR family of genes is known to be highly expressed in NSCLC tumors and play a critical role in the progression of the disease. Although patients initially respond well to EGFR TKI therapy, acquired resistance occurs through several mechanisms, including compensatory cMET pathway activation (Robinson et al. 2013). Previous reports have demonstrated that soluble forms of EGFR, Her2 and cMET are generated through either alternate splicing of mRNA or proteolytic cleavage of the full length receptors (Wilken et al. 2013). Serum concentrations of these receptors can be correlated with prognosis as well as treatment response (Gregorc, 2004, Kasahara, 2010 Heinmoller, 2003). Soluble receptor ligands, including Hepatocyte growth factor (HGF), have also been evaluated in NSCLC and a strong correlation has also been observed between the levels of serum HGF and disease outcome in patients treated with EGFR-TKIs (Kasahara et al. 2010). In this study, we evaluated concentrations of sEGFR, sMET, sHER2,, and HGF in NSCLC versus healthy normal serum samples via meso scale discovery (MSD) platform. Inventoried and custom MSD assays were optimized to measure these proteins in a training set consisting of 19 NSCLC and 16 normal healthy serum samples. Significantly lower concentrations of sEGFR and sHER2 were observed in the NSCLC serum samples compared to normal (p value = 0.0092 for both receptors), whereas, in contrast, the levels of HGF were significantly higher in the NSCLC patient samples (p value Citation Format: Dana S. Gaffney, Katherine Bell, Gabriela Martinez, Jayaprakash Karkera, Suso Platero. Detection of soluble protein biomarkers in NSCLC serum samples by meso scale discovery electrochemiluminescence platform. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5027.

Abstract 3990: FGFR3 mutations as novel oncogenic targets

Fibroblast growth factors (FGFs) are a family of homologous secreted glycoproteins involved in signaling pathways responsible for embryonic development, cell proliferation, survival, and migration. FGF activity is mediated by four transmembrane fibroblast growth factor receptors (FGFRs) which are receptor tyrosine kinases. Typically, FGF binding induces FGFR dimerization, leading to phosphorylation of the intracellular tyrosine kinase domain. This leads to downstream activation of multiple signaling pathways, including the mitogen-activated protein kinase (MAPK), PI3K/AKT, signal transducer and activator of transcription (STAT), and phospholipase-C-γ cascades. Deregulated FGFR activity, through mutations or translocations, is often associated with oncogenic events. Aberrations in FGFR genes have been observed in several tumor types including bladder, gastric, colorectal, ovarian, and hematologic cancers. To date, several FGFR3 mutations have been identified in bladder cancer. In this study, we developed a TaqMan qRT-PCR-based approach to detect four FGFR3 mutations in formalin-fixed paraffin embedded tissue (FFPET) samples. Cell lines overexpressing FGFR3 mutations were generated to determine impact on cell signaling, and sensitivity to the small-molecule pan-FGFR inhibitor JNJ-42756493. To determine the role and significance of these FGFR3 mutations in cancer, mutation expression constructs were designed and individually transfected into normal rat kidney epithelial cells. Cells harboring the FGFR3 mutations exhibited anchorage-independent growth, increased proliferation, and showed increased sensitivity to the FGFR inhibitor JNJ-42756493 in vitro compared to parental lines. These findings underline the oncogenic potential of the FGFR3 mutation genes and highlight their unique potential as predictive biomarkers in the selection of patients for FGFR-targeted therapy. Citation Format: Gabriela Martinez Cardona, Dana Gaffney, Katherine Bell, Joseph Portale, Matthew Dunworth, Matthew Lorenzi, Suso Platero, Jayaprakash Karkera. FGFR3 mutations as novel oncogenic targets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3990.

Abstract 4326: Co-amplification of FGF receptors and ligands in FGFR inhibitor-sensitive cell lines

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The fibroblast growth factor receptors (FGFR) play a key role during development and in adult function. FGFRs belong to a family of receptor tyrosine kinases which are single-pass transmembrane receptors with extracellular ligand binding domains and an intracellular tyrosine kinase domain. Upon binding of the ligand the kinase domain activates intracellular signaling networks that coordinate cellular processes such as proliferation, growth, differentiation, migration and survival. Fibroblast growth factors (FGFs) are a family of 18 ligands which are able to bind and activate distinct FGFRs. Deregulated FGFR activity, through mutations or translocations, is often associated with oncogenic events. Overexpression of FGFR or FGF may lead to increased cell proliferation, growth, differentiation, migration or survival, thus making it an interesting target. In this study we optimized a TaqMan qRT-PCR-based approach to evaluate the copy number variation for several FGFRs (FGFR1, FGFR2, FGFR3 and FGFR4) and several FGF ligands (FGF1, FGF2, FGF3, FGF4, FGF10, FGF12, and FGF19) on a panel of 23 cell lines. The cell types covered a variety of diseases including bladder, breast, endometrial, gastric, kidney, liver, lymphoma, melanoma, sarcoma, small cell lung carcinoma and squamous cell carcinoma. These cell lines had been tested for sensitivity to the JNJ FGFR small molecule inhibitor JNJ42541707 which is a pan-FGFR inhibitor. We observed in the sensitive (IC50<100nm) and moderately sensitive (IC50 100 nM - 1000 nM) cell lines a co-amplification of FGF receptor and ligand. Two of the most sensitive cell lines to FGFR inhibitor treatments had the greatest amplification of FGFR2 with a copy number value greater than 100. This finding that co-amplification occurs in receptor and ligand offers a new potential biomarker for patients that may be sensitive to small molecule FGFR inhibitors. Citation Format: Katherine Bell, Dana Gaffney, Gabriela Martinez-Cardona, Jayaprakash Karkera, Suso Platero. Co-amplification of FGF receptors and ligands in FGFR inhibitor-sensitive cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4326. doi:10.1158/1538-7445.AM2015-4326

Abstract 4325: The role of FGFR fusion genes as novel oncogenic targets

Fusion genes are chromosomal aberrations that are found in many cancers and can be used as prognostic markers and drug targets in clinical practice. Gene fusions can lead to the production of oncogenic fusion proteins or to enhanced expression of oncogenes. Advances in next-generation sequencing technologies have made possible to identify more efficiently novel fusion proteins in cancer. Recently, several FGFR fusion genes with intact kinase domains have been identified in bladder, lung, breast, thyroid, oral, and prostate cancers, as well as other tumor types. To date, several FGFR3 partner genes have been identified. In this study, we focused on the FGFR3-BAIAP2L1 and FGFR3-TACC3 gene fusions and investigated their tumorigenic activity, mechanism of activation, and sensitivity to the FGFR inhibitor JNJ-42756493. To determine the role and significance of FGFR fusion genes in cancer, FGFR fusion expression constructs were designed and individually transfected into normal rat kidney epithelial cells. FGFR fusion overexpressing cells not only showed increased cell proliferation, but also exhibited anchorage-independent cell growth. Cells harboring the FGFR fusions showed increased sensitivity to the FGFR inhibitor JNJ-42756493 in vitro, whereas the wild-type FGFR3 did not in the absence of FGF ligands. In addition, Western blotting analyses indicated that the overexpression of the FGFR fusions resulted in highly activated proteins that induce signaling via the MAPK pathway. These findings underline the oncogenic potential of the FGFR fusion genes and highlight their unique potential as predictive biomarkers in the selection of patients for FGFR-targeted therapy. Citation Format: Gabriela Martinez Cardona, Katherine Bell, Dana Gaffney, Joseph Portale, Suso Platero, Matthew Lorenzi, Jayaprakash Karkera. The role of FGFR fusion genes as novel oncogenic targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4325. doi:10.1158/1538-7445.AM2015-4325

Abstract B03: Identification of R-spondin fusions in NSCLC.

Autocrine Wnt signaling has been reported in a significant fraction of NSCLC. The R-spondin (RSPO) protein family is a small family of four secreted growth factors. The four paralogs share 40–60% pairwise amino acid sequence identity and are predicted to share substantial structural homologies. RSPO proteins are involved in vertebrate development and their ligand-type activities overlap substantially with those of canonical Wnt ligands. A characteristic feature of all four RSPO members is their ability to activate β-catenin signaling and enhance WNT-mediated β-catenin activation. It has recently been described that recurrent gene fusions involving RSPO family members of RSPO2 and RSPO3 that occur in ~10% of colon tumors and are mutually exclusive with other WNT pathway genetic alterations. In this study we developed a TaqMan qRT-PCR-based approach to evaluate systematically the expression of these three (3) RSPO fusion transcripts in formalin-fixed paraffin embedded tissue (FFPET) samples. We examined 324 NSCLC samples that included 197 squamous and 127 adenocarcinoma subtypes for the presence of EIF3E(e1)-RSPO2(e1), PTPRK(e1)-RSPO3(e2), PTPRK(e7)-RSPO3(e2). EIF3E(e1)-RSPO2(e1) was identified in 1% and this fusion transcript is expected to produce a functional RSPO2 protein driven by the EIF3E promoter. The PTPRK(e1)–RSPO3(e2) transcript found in 2% and is an in-frame fusion that preserves the entire coding sequence of RSPO3 and replaces its secretion signal sequence with that of PTPRK. Interestingly, all the fusions were detected only in the squamous subtype of NSCLC. These findings suggest an important role for dysregulated Wnt-β-catenin signaling in lung cancer and identify a new driver segment in NSCLC for therapeutic intervention. Citation Format: Jayaprakash Karkera, Gabriela Martinez, Katherine Bell, Joseph Portale, Dana Gaffney, Matthew V. Lorenzi, Suso Platero. Identification of R-spondin fusions in NSCLC. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B03.

Abstract 1548: Genomic and molecular profiling of NSCLC formalin-fixed paraffin-embedded tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Non-Small Cell Lung Cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 85% of all lung cancers worldwide. Standard therapy options include surgical resection followed by radiation and/or chemotherapy. More recently, analysis of the genomic profile of NSCLC has led to the development of molecularly targeted treatment strategies designed to enhance survival of select subsets of patients containing pre-determined genetic alterations, including gene mutations and aberrant gene expression profiles. In this study, we have developed a TaqMan-based approach to evaluate the genomic profile of 339 NSCLC FFPE tumors, consisting of 152 adenocarcinoma and 187 squamous cell carcinoma (SCC), stages I-IV. Using a custom qRT-PCR somatic mutation array, we have identified the frequencies of key mutations prevalent in NSCLC- associated genes, including EGFR: 5% SCC , 29% adenocarcinoma, cMET: 6% SCC, 4% adenocarcinoma, and KRAS: 3% SCC, 19% adenocarcinoma. Overall, these frequencies are concordant with previous reports. Additionally, we have evaluated the gene expression and IHC profiles of NSCLC-driver signaling pathways including EGFR, cMET and HGF. Various levels of expression for EGFR, cMET and HGF were observed across all samples, with a significant association detected between the presence of EGFR mutation and high EGFR expression in adenocarcinoma (p value = 0.0046). Overall, our findings represent a comprehensive catalog of common genetic aberrations with concurrent gene and protein expression profiles in NSCLC. Furthermore, this data provides insight into correlations observed across these molecular characteristics contributing to NSCLC tumor development. These insights can provide guidance for patient stratification and novel therapeutic strategies for select targeted therapies in NSCLC. Citation Format: Dana S. Gaffney, Katherine Bell, Gabriela Martinez, Yashoda Rajpurohit, Jayaprakash Karkera, Christopher Moy, Suso Platero. Genomic and molecular profiling of NSCLC formalin-fixed paraffin-embedded tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1548. doi:10.1158/1538-7445.AM2014-1548

Abstract 3174: A real-time qPCR approach to detect fusions between the KIF5B and RET genes in non-small cell lung cancer.

Lung cancer is the leading cause of death in cancer worldwide. There are two major forms of lung cancer, including small cell lung cancer (SCLC) which accounts for approximately 20% of all lung cancers and non-small cell lung cancer (NSCLC) which accounts for approximately 80% of lung cancers. Around 25% of these lung cancer patients are never smokers and these cancers tend to be the result of single somatic mutation events. Several somatic events have been reported in NSCLC, including mutations in EGFR and KRAS along with an EML4-ALK fusion gene, however more than 40% of these cancers are the result of unknown genetic events. Recently several papers have reported a novel fusion gene resulting from a 10.6 Mb inversion on chromosome 10 which leads to a fusion between the KIF5B and RET genes. The RET gene is a well-known tyrosine-kinase proto-oncogene which has been linked to papillary thyroid carcinomas and its expression is generally very low in lung. RET tyrosine kinase stimulates autophosphorylation of the tyrosine kinase unit which activates several pathways including STAT3, RAS/ERK, MAPK, PI3K/AKT and SRC. In the KIF5B-RET fusion KIF5B retains its coiled-coil domain necessary for homodimerization and the RET retains its kinase function leading to aberrant activation of several kinase pathways. Several fusion genes between the exons of KIF5B and RET have been previously reported including KIF5B15:RET12, KIF5B16:RET12, KIF5B22:RET12, KIF5B23:RET12, KIF5B15:RET11, KIF5B24:RET8 and KIF5B24:RET11. In this study we developed a TaqMan qRT-PCR-based approach to evaluate the expression of these seven (7) KIF5B-RET fusion transcripts in 64 NSCLC fresh frozen biopsies, ranging from stage I to stage III, including 25 adenocarcinoma and 37 squamous cell carcinoma samples, respectively. Our findings confirm the presence of the fusion between KIF5B15 (exon 15) and RET12 (exon 12) at a frequency of 1.56% in all subtypes. The clinicopathological background of the KIF5B/RET fusion-positive patient agrees with previously reported trends for this fusion event consisting of a caucasian female, non smoker, with adenocarcinoma subtype. Although this percentage is relatively small, it still represents around 12,000 individuals worldwide that express this fusion transcript, presenting a promising biomarker for targeted therapeutics in the treatment of NSCLC disease. Citation Format: Katherine Bell, Dana Gaffney, Gabriela Martinez, Suso Platero, Jayaprakash Karkera. A real-time qPCR approach to detect fusions between the KIF5B and RET genes in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3174. doi:10.1158/1538-7445.AM2013-3174