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.

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

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.

Background: The molecular apocrine (MA) subtype of breast cancer is identified by gene expression profiling. MA tumors are estrogen receptor (ER) negative and progesterone receptor (PR) negative, but still express estrogen responsive genes. The androgen receptor (AR) pathway may be driving growth in these tumors because androgen responsive genes are expressed in tumors with the MA gene signature. The MA gene signature is identified in approximately 10% of triple negative breast cancer (TNBC) and may predict patients with tumors responsive to agents that inhibit the AR pathway. AR protein expression, measured by immunohistochemistry (IHC), may be a surrogate for the MA gene signature, but to date, a careful comparison of gene expression profiles and AR protein expression has not been conducted. In this study, cohorts of TNBCs were assessed for the MA gene signature and these results were compared with AR IHC expression and with a novel gene expression assay that may predict tumors with the MA gene signature. Methods: Formalin fixed, paraffin-embedded (FFPE) TNBC samples were commercially obtained. ER, PR and HER2 status of these samples was confirmed by IHC. AR expression was detected by IHC using two different antibody clones. Both staining intensity and percent positive cells were recorded for each sample. Gene expression data was collected from a cohort of TNBC FFPE samples using cDNA-mediated Annealing, Selection, extension, and Ligation (DASL) technology. A 2-gene classifier of the MA gene expression signature was derived by interrogating publically available gene expression data from ER-negative breast cancers. A reverse-transcriptase polymerase chain reaction (RT-PCR) assay to detect the 2-gene classifier was developed. Cell lines predicted to have the MA gene signature by the 2-gene assay were tested for sensitivity to R-1881 in vitro. Results: Using computational approaches and publically available datasets, we confirmed the validity of the MA gene signature and estimated the prevalence to be between 12% and 37% in ER-negative breast tumors. The 2-gene classifier was 100% specific in determining MA tumors in a training set using gene expression data as a standard. In a validation set, the 2-gene assay was 66% correlative with AR IHC positivity when the IHC cut-off was set at 10% positive tumor cells. Cell lines predicted to express the MA gene signature by the 2-gene classifier proliferated in response to androgen. This effect was blocked by Flutamide. Conclusions: These results indicate that AR IHC using a 10% cut-off may not completely correlate with the MA gene signature. Further refinement of AR IHC scoring criteria may produce greater specificity. Cell proliferation data suggests the 2-gene assay can predict tumors that will proliferate in response to androgen. Work is ongoing to determine the correlation between the 2-gene assay results, AR IHC and DASL gene expression data to fully understand the predictability of this assay. Understanding this correlation may allow use of simple clinical assays to accurately select patients responsive to agents that block AR signaling. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-01-09.

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

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

Many advances have been made in the diagnosis and treatment of breast cancer in recent years, but it still remains one of the leading causes of death in women. It is well known that the growth of breast cells is regulated by the interactions of different steroid hormones, in particular androgens and estrogens that are related due to their connected metabolic pathways. A number of novel inhibitors of steroidogenic enzymes have been developed that target pre-receptor events_those pertaining to the production, transport, and conversion of steroid ligands. Critical pre-receptor steps include the conversion of pregnenolone-like steroids into androgens, mediated largely by the 17α-hydroxylase/lyase (CYP17) enzyme complex, and the conversion of testosterone and androstenedione to estradiol and estrone, mediated by aromatase (CYP19). Both conversions are implicated in the emergence of tumor resistance and thus are targets for intervention. Androgen Receptor (AR) is the sex hormone receptor most frequently found in both primary and secondary breast tumors, which is indicitative of the importance of AR in regulating the growth of breast cancer cells. It is estimated that 90% of human genes undergo alternative splicing and AR is no exception. Alternative splicing of the AR could culminate in a receptor that is capable of translocation, or can bind DNA without ligand, leading current AR therapies to be less efficacious. Using TaqMan qRT-PCR we examined 213 female breast-cancer FFPET samples, 80 ER- PR- Her2- samples, 68 ER- PR- Her2+ samples, and 64 ER+ PR+ Her2- samples, as well as 8 breast-cancer cell lines for the presence of ESR1, CYP17, CYP19, full length AR and AR splice variants ARV1, ARV3/V7, ARV567, and Delta3AR. ARV3/V7 and Delta3AR were the most prevalent variants in the ER+ PR+ Her2- and ER- PR- Her2+ sample sets, with >85% of these samples showing expression of either or both of these variants. On the other hand, ARV1, ARV3/V7, and ARV567 were the most prevalent variants in the ER- PR- Her2- sample set, with >90% of these samples showing expression of one or a combination of these variants. Lower expression values of most of the AR variants were observed in higher grade ER+ PR+ Her2- and ER- PR- Her2+ samples as compared to the lower grade samples. CYP19 was highly prevalent in all sample sets with >75% of all samples showing expression, while CYP17 expression was observed in <30% of all the samples tested. Our findings show the relatively high expression of AR variants and CYP19 in breast cancer tissues, which may indicate their role in regulating the growth of these tumors. Hence increased expression of AR splice variants in breast cancer tumors may be an important biomarker of resistance and targets for AR related therapy. Citation Format: Gabriela Martinez, Dana Gaffney, Katherine Bell, Suso Platero, Deborah Ricci, Weimin Li, Jayaprakash D. Karkera. Identification of androgen receptor splice variants, ESR1, CYP17, and CYP19 in human breast 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 3467. doi:10.1158/1538-7445.AM2013-3467

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