Dennis Peters

Identification of recurrent FGFR3 fusion genes in lung cancer through kinome‐centred RNA sequencing

Oncogenic fusion genes that involve kinases have proven to be effective targets for therapy in a wide range of cancers. Unfortunately, the diagnostic approaches required to identify these events are struggling to keep pace with the diverse array of genetic alterations that occur in cancer. Diagnostic screening in solid tumours is particularly challenging, as many fusion genes occur with a low frequency. To overcome these limitations, we developed a capture enrichment strategy to enable high‐throughput transcript sequencing of the human kinome. This approach provides a global overview of kinase fusion events, irrespective of the identity of the fusion partner. To demonstrate the utility of this system, we profiled 100 non‐small cell lung cancers and identified numerous genetic alterations impacting fibroblast growth factor receptor 3 (FGFR3) in lung squamous cell carcinoma and a novel ALK fusion partner in lung adenocarcinoma.

ERBB2 Mutations Characterize a Subgroup of Muscle-invasive Bladder Cancers with Excellent Response to Neoadjuvant Chemotherapy

UNLABELLED A pathologic complete response to neoadjuvant chemotherapy (NAC) containing platinum is a strong prognostic determinant for patients with muscle-invasive bladder cancer (MIBC). Despite comprehensive molecular characterization of bladder cancer, associations of molecular alterations with treatment response are still largely unknown. We selected pathologic complete responders (ypT0N0; n=38) and nonresponders (higher than ypT2; n=33) from a cohort of high-grade MIBC patients treated with NAC. DNA was isolated from prechemotherapy tumor tissue and used for next-generation sequencing of 178 cancer-associated genes (discovery cohort) or targeted sequencing (validation cohort). We found that 9 of 38 complete responders had erb-b2 receptor tyrosine kinase 2 (ERBB2) missense mutations, whereas none of 33 nonresponders had ERBB2 mutations (p=0.003). ERBB2 missense mutations in complete responders were mostly confirmed activating mutations. ERCC2 missense mutations, recently found associated with response to NAC, were more common in complete responders; however, this association did not reach statistical significance in our cohort. We conclude that ERBB2 missense mutations characterize a subgroup of MIBC patients with an excellent response to NAC. PATIENT SUMMARY In this report we looked for genetic alterations that can predict the response to neoadjuvant chemotherapy (NAC) in bladder cancer. We found that mutations in the gene ERBB2 are exclusively present in patients responding to NAC.

Transcription Factor NFIB Is a Driver of Small Cell Lung Cancer Progression in Mice and Marks Metastatic Disease in Patients

Summary Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor, and no effective treatment is available to date. Mouse models of SCLC based on the inactivation of Rb1 and Trp53 show frequent amplifications of the Nfib and Mycl genes. Here, we report that, although overexpression of either transcription factor accelerates tumor growth, NFIB specifically promotes metastatic spread. High NFIB levels are associated with expansive growth of a poorly differentiated and almost exclusively E-cadherin (CDH1)-negative invasive tumor cell population. Consistent with the mouse data, we find that NFIB is overexpressed in almost all tested human metastatic high-grade neuroendocrine lung tumors, warranting further assessment of NFIB as a tumor progression marker in a clinical setting.

Tumor heterogeneity of fibroblast growth factor receptor 3 (FGFR3) mutations in invasive bladder cancer: implications for perioperative anti-FGFR3 treatment

BACKGROUND Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer. Preclinical studies show that anti-FGFR3 treatment slows down tumor growth, suggesting that this tyrosine kinase receptor is a candidate for personalized bladder cancer treatment, particularly in patients with mutated FGFR3. We addressed tumor heterogeneity in a large multicenter, multi-laboratory study, as this may have significant impact on therapeutic response. PATIENTS AND METHODS We evaluated possible FGFR3 heterogeneity by the PCR-SNaPshot method in the superficial and deep compartments of tumors obtained by transurethral resection (TUR, n = 61) and in radical cystectomy (RC, n = 614) specimens and corresponding cancer-positive lymph nodes (LN+, n = 201). RESULTS We found FGFR3 mutations in 13/34 (38%) T1 and 8/27 (30%) ≥T2-TUR samples, with 100% concordance between superficial and deeper parts in T1-TUR samples. Of eight FGFR3 mutant ≥T2-TUR samples, only 4 (50%) displayed the mutation in the deeper part. We found 67/614 (11%) FGFR3 mutations in RC specimens. FGFR3 mutation was associated with pN0 (P < 0.001) at RC. In 10/201 (5%) LN+, an FGFR3 mutation was found, all concordant with the corresponding RC specimen. In the remaining 191 cases, RC and LN+ were both wild type. CONCLUSIONS FGFR3 mutation status seems promising to guide decision-making on adjuvant anti-FGFR3 therapy as it appeared homogeneous in RC and LN+. Based on the results of TUR, the deep part of the tumor needs to be assessed if neoadjuvant anti-FGFR3 treatment is considered. We conclude that studies on the heterogeneity of actionable molecular targets should precede clinical trials with these drugs in the perioperative setting.

FGFR1, 2 and 3 protein overexpression and molecular aberrations of FGFR3 in early stage non‐small cell lung cancer

This study aimed to determine protein expression levels of fibroblast growth factor receptors (FGFR) 1, 2 and 3 in early stage non‐small cell lung cancer (NSCLC). Additionally, a screen to define the frequency of FGFR3‐TACC3 translocation and FGFR3 amplification was performed. Archived tissues from 653 NSCLC samples (adenocarcinoma (AC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC)) were analysed with immunohistochemistry (IHC) for expression of FGFR1, 2 and 3. Expression levels of FGFR1, 2 and 3 were correlated with clinicopathological features. The presence of FGFR3‐TACC3 translocation was detected by RT‐PCR and FGFR3 amplification was detected by fluorescence in situ hybridization. FGFR1, 2 and 3 proteins were highly expressed in 64 (10.6%), 76 (12.9%) and 20 (3.3%) NSCLC tumour samples, respectively. Protein expression of FGFR1 was significantly related to worse overall survival in NSCLC. Furthermore, FGFR1 protein expression was associated with light smoking and histological subtype (AC), FGFR2 protein expression with female gender, younger age, histological subtype (AC) and lower tumour stage, and FGFR3 protein was significantly overexpressed in tumours of older patients and SCC histology. The FGFR3‐TACC3 fusion was detected in 3.0% (6/200) of NSCLC samples and the FGFR3 gene was amplified in 4.7% of IHC positive NSCLC samples (2/43). FGFR1, 2 and 3 proteins are expressed in a high number of early stage NSCLC and FGFR1 protein expression may serve as a prognostic biomarker. Recurrent translocations and amplifications in FGFR3 can be found in NSCLC. This study shows that FGFR family members are frequently aberrant in NSCLC and could be interesting therapeutic targets for the treatment of NSCLC.

mTOR pathway activation is a favorable prognostic factor in human prostate adenocarcinoma

Prostate cancer patients with localized disease are treated with curative intent. However, the disease will recur in approximately 30% of patients with a high incidence of morbidity and mortality. Prognostic biomarkers are needed to identify patients with high risk of relapse. mTOR pathway activation is reported in prostate cancer, but clinical trials testing efficacy of mTOR inhibitors were unsuccessful. To explain this clinical observation, we studied the expression and prognostic impact of mTOR-S2448 phosphorylation in localized prostate carcinomas. mTOR-S2448 phosphorylation is indicative for an activated mTOR pathway in prostate cancer. Surprisingly, the mTOR signaling pathway is activated specifically in prostate cancer patients with a favorable outcome. Since tumors from poor-outcome patients have low levels of mTOR-S2448 phosphorylation, this may explain why mTOR inhibitors proved unsuccessful in prostate cancer trials.

Assessment of PD-L1 expression across breast cancer molecular subtypes, in relation to mutation rate, BRCA1-like status, tumor-infiltrating immune cells and survival

ABSTRACT To better understand the expression pattern of programmed death-ligand 1 (PD-L1) expression in different breast cancer types, we characterized PD-L1 expression in tumor and tumor-infiltrating immune cells, in relation to mutation rate, BRCA1-like status and survival. We analyzed 410 primary treatment-naive breast tumors comprising 162 estrogen receptor-positive (ER+) and HER2−, 101 HER2+ and 147 triple-negative (TN) cancers. Pathologists quantified tumor-infiltrating lymphocytes (TILs) and PD-L1 expression in tumor cells and TILs using whole slides and tissue microarray. Mutation rate was assessed by DNA sequencing, BRCA1-like status using multiplex ligation-dependent probe amplification, and immune landscape by multiplex image analyses of CD4, CD68, CD8, FOXP3, cytokeratin, and PD-L1. Half of PD-L1 scores evaluated by tissue microarray were false negatives compared to whole slide evaluations. We observed at least 1% of PD-L1-positive (PD-L1+) cells in 53.1% of ER+HER2−, 73.3% of HER2+, and 84.4% of TN tumors. PD-L1 expression was higher in ductal compared to lobular carcinomas, also within ER+HER2− tumors (p = 0.04). High PD-L1+ TILs score (> 50%) was independently associated with better outcome in TN tumors (HR = 0.27; 95%CI = 0.10–0.69). Within TN tumors, PD-L1 and TIL scores showed a modest but significant positive association with the number of silent mutations, but no association with BRCA1-like status. Multiplex image analyses indicated that PD-L1 is expressed on multiple immune cells (CD68+ macrophages, CD4+, FOXP3+, and CD8+ T cells) in the breast tumor microenvironment, independent of the PD-L1 status of the tumor cells. We found no evidence that levels of PD-L1+ TILs in TN breast cancer are driven by high mutation rate or BRCA1-like status.

Trophoblast Glycoprotein is Associated With a Favorable Outcome for Mesothelioma and a Target for Antibody Drug Conjugates

Introduction: The prognosis for patients with mesothelioma is poor, which prompts the need for the development of better treatment options. Antibody drug conjugates (ADCs) are gaining interest as a therapeutic strategy in mesothelioma. Trophoblast glycoprotein (5T4) is an oncofetal protein overexpressed in mesothelioma with low expression in normal tissue and therefore a good candidate for ADC treatment. Here, we evaluated and manipulated 5T4 as a suitable antigen for ADC targeted therapy in patients with mesothelioma. Methods: Expression of the 5T4 antigen is evaluated in (primary) mesothelioma cell lines and biopsy specimens, and correlated with clinical outcome. Internalization was assessed in 5T4 expressing cells. The cytotoxicity of three different 5T4‐targeting ADCs was tested on (primary) mesothelioma cells. Results: 5T4 was expressed in 10 of 12 (primary) cell lines. Most biopsy specimens stained positive for the 5T4 antigen, with marked differences in staining intensity and percentage of positive cells. High expression correlated with long progression‐free survival. Both free antibody and ADCs targeting 5T4 were internalized and entered lysosomal compartments. Cytotoxicity experiments showed that cell lines with a high expression for 5T4 were sensitive to two of three ADCs. Lack of efficacy for the third ADC could be restored by neutralizing lysosomal compartments with chloroquine. Conclusions: The 5T4 antigen is expressed in mesothelioma and 5T4‐based ADCs are internalized in lysosomes. Two of three ADCs were capable of killing the mesothelioma cells; the third ADC required additional lysosomal neutralization for its effect. 5T4‐based ADCs would be a selective strategy for the treatment of mesothelioma.

Abstract 575: PD-L1 positive tumor-infiltrating lymphocytes and mutational load in breast cancer

Background: PD-1 blockade has emerged as an effective treatment for a subset of cancer patients. Studies have shown that PD-L1 expression is associated with likelihood of response to PD-1 blockade. In order to select the right breast cancer patient for immunotherapy, characterization of the immune landscape of breast tumors is required. Therefore, we assessed PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in different breast tumor subtypes and the link with prognosis. We also sequenced a panel of genes to assess the mutational load in triple negative tumors (TNBC) and investigate the association with PD-L1 positive TILs. Material and methods: We analyzed 438 tumor samples from breast cancer patients of all ages treated between 1986 and 2007 with surgery, with or without adjuvant therapy. PD-L1 was stained using whole slide specimens (E1L3N® antibody) after methodological validation. Pathologists quantified TILs based on International TILs Working Group recommendations and scored PD-L1 based on the percentage of positive (tumor and/or immune) cells; as negative if 0%, positive if ≥1%, and high if >50%. Mutational load was assessed based on DNA kinome sequencing. Associations were measured by Cox/logistic regression model, including pathological variables. Multiplex imaging of 20 immune-infiltrated areas from four ER negative tumors were performed using the Vectra® system based on immunofluorescence staining panel of: CD4, CD68, CD8, FOXP3 and PD-L1. Results: PD-L1 expression and TILs were higher in ductal (compared with lobular), high grade and estrogen receptor (ER)-negative tumors (p 50%) was significantly associated with better DMFS: HR=0.51; 95%CI: 0.27-0.98. TNBC with high PD-L1 expression of TILs (>50%) showed an association with increased mutation load (p=0.019) and a trend for better DMFS (HR=0.41; 95%CI: 0.16-1.04) compared with tumors lacking TILs. Further characterization of PD-L1 positivity in the immune-infiltrated cells was conducted by a multiplex imaging analysis. Preliminary results indicated that PD-L1 is expressed in CD68+, CD4+, FOXP3+ and CD8+ immune-cells. Conclusion: Our findings suggest that PD-L1 positive TILs are associated with worse prognosis in ER-positive breast cancer and with better outcome in ER-negative group. In TNBC, high mutational load correlates with high PD-L1 positive TILs. Citation Format: Marcelo Sobral-Leite, Koen Van de Vijver, Magali Michaut, Hugo M. Horlings, Tesa M. Severson, Philip C. Schouten, Rianne van der Linden, Kelly Kersten, Anna Marie Mulligan, Nayana Weerasooriya, Joyce Sanders, Ashley Cimino-Mathews, Dennis Peters, Gerrit K. Hooijer, Erik Hooijberg, Annegien Broeks, Rene Bernards, Sabine Linn, Irene L. Andrulis, Marc J. van de Vijver, Lodewyk F. Wessels, Marleen Kok, Karin E. de Visser, Marjanka K. Schmidt. PD-L1 positive tumor-infiltrating lymphocytes and mutational load in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 575. doi:10.1158/1538-7445.AM2017-575

MP28-20 PATHOLOGICAL VALIDATION OF ADJUVANT ANTI - FIBROBLAST GROWTH FACTOR RECEPTOR 3 (FGFR3) FOR PERSONALIZED TREATMENT OF BLADDER CANCER

Yann NEUZILLET*, Laura MERTENS, Amsterdam, Netherlands; Shahrokh SHARIAT, Dallas, TX; Peter BOSTROM, Tuomas MIRTTI, Turku, Finland; Arthur SAGALOWSKY, Raheela ASHFAQ, Dallas, TX; Annegien BROEKS, Michiel VAN, DER HEIJDEN, Dennis PETERS, Chantall CURIAL, Jeroen DE JONG, Simon HORENBLAS, Amsterdam, Netherlands; Carolyn HURST, Darren TOMLINSON, Margaret KNOWLES, Leeds, United Kingdom; Bharati BAPAT, Michael JEWETT, Alexandre ZLOTTA, Toronto, Canada; Joyce SANDERS, Amsterdam, Netherlands; Yair LOTAN, Dallas, TX; Theo VAN DER KWAST, Toronto, Canada; Bas VAN RHIJN, Amsterdam, Netherlands