Jeroen de Jong

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.

Additional weekly Cetuximab to concurrent chemoradiotherapy in locally advanced non-small cell lung carcinoma: efficacy and safety outcomes of a randomized, multi-center phase II study investigating.

BACKGROUND Modest benefits from concurrent chemoradiotherapy in patients with locally advanced NSCLC warrant further clinical investigations to identify more effective treatment regimens. Cetuximab, a monoclonal antibody against the epidermal growth factor receptor has shown activity in NSCLC. We report on the safety and efficacy of the combination of daily dose Cisplatin and concurrent radiotherapy with or without weekly Cetuximab. PATIENTS AND METHODS Patients received high dose accelerated radiotherapy (66 Gy in 24 fractions) and concurrent daily Cisplatin (6 mg/m(2)) without (Arm A) or with (Arm B) weekly Cetuximab (400 mg/m(2) loading dose one week prior to radiotherapy followed by weekly 250 mg/m(2)). The primary endpoint of the trial was objective local control rate (OLCR) determined at 6-8 weeks after treatment. Toxicity was reported as well. RESULTS Between February 2009 and May 2011, 102 patients were randomized. Median follow up was 29 months. The OLCR was 84% in Arm A and 92% in Arm B (p=0.36). The one-year local progression free interval (LPFI) and overall survival (OS) were 69% and 82% for Arm A and 73% and 71% for Arm B, respectively (LPFI p=0.39; OS p=0.99). Toxicity compared equally between both groups. CONCLUSION The addition of Cetuximab to radiotherapy and concurrent Cisplatin did not improve disease control in patients with locally advanced NSCLC but increased treatment related toxicity.

Expression of Programmed Death Ligand 1 in Penile Cancer is of Prognostic Value and Associated with HPV Status

Purpose: PD‐L1 (programmed death ligand 1) inhibits T‐cell function and prevents tumor eradication. This is facilitated by PD‐L1 positive tumor cells and PD‐L1 positive immune cells, and can be prevented by anti‐PD‐1 (programmed death 1)/PD‐L1 immunotherapy. In advanced penile cancer there is a need for new therapeutic strategies. We investigated PD‐L1 expression in penile cancers and compared PD‐L1 expression with disease specific survival, lymph node metastases at diagnosis and high risk HPV status in a large patient cohort. Materials and Methods: A total of 213 primary tumors were immunohistochemically stained for PD‐L1 and scored for tumor (percentage), stroma (binary) and PD‐L1 positive tumor infiltrating macrophages. Additionally, PD‐L1 positive tumors were scored for expression pattern, that is diffuse or predominantly present at the tumor‐stroma margin. Results: Staining was successful in 200 tumors, of which 75% were high risk HPV negative. Median followup was 62 months. Of 200 tumors 96 (48%) were PD‐L1 positive (scored 1% or greater), of which 59 (62%) had a marginal expression pattern and 79 (82%) were high risk HPV negative (p = 0.03). Compared to PD‐L1 negative tumors, the PD‐L1 expression patterns had different prognostic values in the whole cohort as well as in the high risk HPV negative subgroup. On multivariable analyses a marginal expression pattern was associated with absent lymph node metastases (OR 0.4) while diffuse expression was associated with poor survival (HR 2.58). These results were more prominent in the high risk HPV negative subgroup (OR 0.25, HR 3.92). Conclusions: PD‐L1 was expressed in 48% of penile carcinomas and mainly in high risk HPV negative tumors. The pattern of expression was a prognostic factor as marginal expression was associated with absent lymph node metastases and diffuse expression was associated with poor survival.

Gross tumor volume and clinical target volume in prostate cancer: How do satellites relate to the index lesion

PURPOSE There is an increasing interest for dose differentiation in prostate radiotherapy. The purpose of our study was to analyze the spatial distribution of tumor satellites inside the prostate. METHODS AND MATERIALS 61 prostatectomy specimens were stained with H&E. Tumor regions were delineated by the uro-pathologist. Volumes, distances and cell densities of all delineated tumor regions were measured and further analyzed. RESULTS Multifocal disease was seen in 84% of the patients. The median number of tumor foci was 3. The median distance between the index lesion and the satellites was 1.0 cm, with a maximum of 4.4 cm. The index lesions accounted for 88% of the total tumor volume. The contribution of tumor foci<0.1 cm(3) to the total tumor volume was 2%. The median cell density of the index lesion and all satellites, regardless of size, were significantly higher than that of the prostate. CONCLUSIONS Satellites do not appear in a limited margin around the index lesion (GTV). Consequently, a fixed CTV margin would not effectively cover all satellites. Thus if the aim is to treat all tumor foci, the entire prostate gland should be considered CTV.

Androgen receptor DNA binding and chromatin accessibility profiling in prostate cancer

Prostate cancer (PCa) is the second most common cancer in men. The Androgen Receptor (AR) is the major driver of PCa and the main target of therapy in the advanced setting. AR is a nuclear receptor that binds the chromatin and regulates transcription of genes involved in cancer cell proliferation and survival. In a study by Stelloo et al. (1) we explored prostate cancer on the level of transcriptional regulation by means of Formaldehyde-Assisted Isolation of Regulatory Elements and Chromatin Immunoprecipitation coupled with massive parallel sequencing (FAIRE-seq and ChIP-seq, respectively). We employed these data for the assessment of differences in transcriptional regulation at distinct stages of PCa progression and to construct a prognostic gene expression classifier. Genomics data includes FAIRE-seq data from normal prostate tissue as well as primary, hormone therapy resistant and metastatic PCa. Furthermore, ChIP-seq data from primary and resistant PCa were generated, along with multiple input controls. The data are publicly available through NCBI GEO database with accession number GSE65478. Here we describe the genomics and clinical data in detail and provide comparative analysis of FAIRE-seq and ChIP-seq data.

Multi-center validation of prostate tumor localization using multi-parametric MRI and prior knowledge.

Purpose: Tumor localization provides crucial information for radiotherapy dose differentiation treatments, such as focal dose escalation and dose painting by numbers, which aim at achieving tumor control with minimal side effects. Multiparametric (mp‐)MRI is increasingly used for tumor detection and localization in prostate because of its ability to visualize tissue structure and to reveal tumor characteristics. However, it can be challenging to distinguish cancer, particularly in the transition zone. In this study, we enhance the performance of a mp‐MRI‐based tumor localization model by incorporating prior knowledge from two sources: a population‐based tumor probability atlas and patient‐specific biopsy examination results. This information typically would be considered by a physician when carrying out a manual tumor delineation. Materials and methods: Our study involves 40 patients from two centers: 23 patients from the University Hospital Leuven (Leuven), Leuven, Belgium and 17 patients from the Netherlands Cancer Institute (NKI), Amsterdam, the Netherlands. All patients received a mp‐MRI exam consisting of a T2‐weighted, diffusion‐weighted, and dynamic contrast‐enhanced MRI before prostatectomy. Thirty‐one features were extracted for each voxel in the prostate. Among these, 29 were from the multiparametric‐MRI, one was from the population‐based tumor probability atlas and one from the biopsy map. T2‐weighted images of each patient were registered to whole‐mount section pathology slices to obtain the ground truth. The study was validated in two settings: single‐center (training and test sets were from the same cohort); and cross‐center (training and test sets were from different cohorts). In addition, automatic delineations created by our model were compared with manual tumor delineations done by six different teams on a subset of Leuven cohort including 15 patients. Results: In the single‐center setting, mp‐MRI‐based features yielded area under the ROC curves (AUC) of 0.690 on a pooled set of patients from both cohorts. Including prevalence into mp‐MRI‐based features increased the AUC to 0.751 and including all features achieved the best performance with AUC of 0.775. Using all features always showed better results when varying the size of the training set. In addition, its performance is comparable with the average performance of six teams delineating the tumors manually. The error rate using all features was 0.22. The two prior knowledge features ranked among the top four most important features out of the 31 features. In the cross‐center setting, combining all features also yielded the best performance in terms of the mean AUC of 0.777 on the pooled set of patients from both cohorts. In addition, the difference in performance between the single‐center setting and cross‐center setting was not significant. Conclusions: The results showed significant improvements when including prior knowledge features in addition to mp‐MRI‐based features in both single‐ and cross‐center settings.

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.

Loss of androgen receptor signaling in prostate cancer‐associated fibroblasts (CAFs) promotes CCL2‐ and CXCL8‐mediated cancer cell migration

Fibroblasts are abundantly present in the prostate tumor microenvironment (TME), including cancer‐associated fibroblasts (CAFs) which play a key role in cancer development. Androgen receptor (AR) signaling is the main driver of prostate cancer (PCa) progression, and stromal cells in the TME also express AR. High‐grade tumor and poor clinical outcome are associated with low AR expression in the TME, which suggests a protective role of AR signaling in the stroma against PCa development. However, the mechanism of this relation is not clear. In this study, we isolated AR‐expressing CAF‐like cells. Testosterone (R1881) exposure did not affect CAF‐like cell morphology, proliferation, or motility. PCa cell growth was not affected by culturing in medium from R1881‐exposed CAF‐like cells; however, migration of PCa cells was inhibited. AR chromatin immune precipitation sequencing (ChIP‐seq) was performed and motif search suggested that AR in CAF‐like cells bound the chromatin through AP‐1‐elements upon R1881 exposure, inducing enhancer‐mediated AR chromatin interactions. The vast majority of chromatin binding sites in CAF‐like cells were unique and not shared with AR sites observed in PCa cell lines or tumors. AR signaling in CAF‐like cells decreased expression of multiple cytokines; most notably CCL2 and CXCL8 and both cytokines increased migration of PCa cells. These results suggest direct paracrine regulation of PCa cell migration by CAFs through AR signaling.

Pathogenic and targetable genetic alterations in 70 urachal adenocarcinomas: Genetic alterations in 70 urachal adenocarcinomas

Urachal cancer (UrC) is a rare but aggressive malignancy often diagnosed in advanced stages requiring systemic treatment. Although cytotoxic chemotherapy is of limited effectiveness, prospective clinical studies can hardly be conducted. Targeted therapeutic treatment approaches and potentially immunotherapy based on a biological rationale may provide an alternative strategy. We therefore subjected 70 urachal adenocarcinomas to targeted next‐generation sequencing, conducted in situ and immunohistochemical analyses (including PD‐L1 and DNA mismatch repair proteins [MMR]) and evaluated the microsatellite instability (MSI) status. The analytical findings were correlated with clinicopathological and outcome data and Kaplan‐Meier and univariable/multivariable Cox regression analyses were performed. The patients had a mean age of 50 years, 66% were male and a 5‐year overall survival (OS) of 58% and recurrence‐free survival (RFS) of 45% was detected. Sequence variations were observed in TP53 (66%), KRAS (21%), BRAF (4%), PIK3CA (4%), FGFR1 (1%), MET (1%), NRAS (1%), and PDGFRA (1%). Gene amplifications were found in EGFR (5%), ERBB2 (2%), and MET (2%). We detected no evidence of MMR‐deficiency (MMR‐d)/MSI‐high (MSI‐h), whereas 10 of 63 cases (16%) expressed PD‐L1. Therefore, anti‐PD‐1/PD‐L1 immunotherapy approaches might be tested in UrC. Importantly, we found aberrations in intracellular signal transduction pathways (RAS/RAF/PI3K) in 31% of UrCs with potential implications for anti‐EGFR therapy. Less frequent potentially actionable genetic alterations were additionally detected in ERBB2 (HER2), MET, FGFR1, and PDGFRA. The molecular profile strengthens the notion that UrC is a distinct entity on the genomic level with closer resemblance to colorectal than to bladder cancer.