Gerrit K.J. Hooijer

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.

Determining sensitivity and specificity of HER2 testing in breast cancer using a tissue micro-array approach

IntroductionOverexpression of the human epidermal growth factor receptor 2 (HER2) as a result of HER2 gene amplification is associated with a relatively poor prognosis in breast cancer and is predictive of HER2-targeting therapy response. False-positive rates of up to 20% for HER2 testing have been described. HER2-testing laboratories are therefore encouraged to participate in external quality control schemes in order to improve HER2-testing standardization.MethodsThis study investigated the feasibility of retesting large numbers of invasive breast cancers for HER2 status on tissue micro-array (TMA) as part of a quality control scheme. For this assessment different HER2 testing methods were used including HER2 detecting antibodies SP3, 4B5, Herceptest and mono color silver in situ hybridization (SISH) and dual color SISH. Final HER2 status for each tumor on the TMA was compared to the local testing result for the same tumor. Discordances between these two results were investigated further by staining whole tumor sections.ResultsFor this study, 1,210 invasive breast carcinomas of patients treated in six hospitals between 2006 and 2008 were evaluated. Results from the three immunohistochemistry (IHC) and two in situ hybridization (ISH) assays performed on the TMAs were compared. The final HER2 status on TMA was determined with SP3, 4B5 and mono color SISH. Concordance between local HER2 test results and TMA retesting was 98.0%. Discordant results between local and TMA retesting were found in 20 tumors (2.0%). False positive HER2 IHC results were identified in 13 (1.3%) tumors; false negative IHC results in seven (0.7%) tumors.ConclusionsRetesting large volumes of HER2 classified breast carcinomas was found to be feasible and can be reliably performed by staining TMAs with SP3, 4B5 and mono color SISH in combination with full-sized slides for discordant cases. The frequency of false-positive results was lower than previously reported in the literature. This method is now offered to other HER2-testing laboratories.

Practical and Robust Identification of Molecular Subtypes in Colorectal Cancer by Immunohistochemistry.

Purpose: Recent transcriptomic analyses have identified four distinct molecular subtypes of colorectal cancer with evident clinical relevance. However, the requirement for sufficient quantities of bulk tumor and difficulties in obtaining high-quality genome-wide transcriptome data from formalin-fixed paraffin-embedded tissue are obstacles toward widespread adoption of this taxonomy. Here, we develop an immunohistochemistry-based classifier to validate the prognostic and predictive value of molecular colorectal cancer subtyping in a multicenter study. Experimental Design: Tissue microarrays from 1,076 patients with colorectal cancer from four different cohorts were stained for five markers (CDX2, FRMD6, HTR2B, ZEB1, and KER) by immunohistochemistry and assessed for microsatellite instability. An automated classification system was trained on one cohort using quantitative image analysis or semiquantitative pathologist scoring of the cores as input and applied to three independent clinical cohorts. Results: This classifier demonstrated 87% concordance with the gold-standard transcriptome-based classification. Application to three validation datasets confirmed the poor prognosis of the mesenchymal-like molecular colorectal cancer subtype. In addition, retrospective analysis demonstrated the benefit of adding cetuximab to bevacizumab and chemotherapy in patients with RAS wild-type metastatic cancers of the canonical epithelial-like subtypes. Conclusions: This study shows that a practical and robust immunohistochemical assay can be employed to identify molecular colorectal cancer subtypes and uncover subtype-specific therapeutic benefit. Finally, the described tool is available online for rapid classification of colorectal cancer samples, both in the format of an automated image analysis pipeline to score tumor core staining, and as a classifier based on semiquantitative pathology scoring. Clin Cancer Res; 23(2); 387–98. ©2016 AACR.

HER2 gene amplification in patients with breast cancer with equivocal IHC results

Aims Equivocal human epidermal growth factor receptor 2 protein (HER2) (2+) immunohistochemistry (IHC) is subject to significant interobserver variation and poses a challenge in obtaining a definitive positive or negative test result. This equivocal test result group accounts for approximately 15% of all tumours, and for optimal guidance of HER2 targeted therapy, a further analysis of quantification of gene copy number and amplification status is needed for patients with early or metastatic breast cancer. Methods 553 breast-cancer specimens with equivocal HER2 IHC(2+) test results were collected and subsequently centrally retested by chromogenic in situ hybridisation (CISH), and HER2 gene copy numbers per tumour cell nucleus were determined. Results Using CISH, 77 of 553 equivocal HER2 IHC(2+) test result cases (13.9% of total) showed high levels of HER2 gene amplification (≥10.0 gene copies per nucleus), and 41 of 553 (7.4% of total) showed low-level HER2 gene amplification (6.0–9.9 gene copies per nucleus). In 73.6% of cases, no amplification of the HER2 gene was shown, and in only 4.9% of cases was an equivocal test result by CISH observed (4.0–5.9 gene copies per nucleus). Conclusions Testing by CISH of all equivocal HER2 IHC(2+) test result provides a definitive guidance in HER2 targeted therapy in 95.1% of cases. A significant proportion (21.3%) of patients with equivocal IHC(2+) test results show amplification of the HER2 gene.

Reliability of core needle biopsy for determining ER and HER2 status in breast cancer

BACKGROUND Several studies have assessed the concordance of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status between core needle biopsy (CNB) and resection specimens, usually in small patient series and with discordant results. PATIENTS AND METHODS ER and HER2 status determined on CNB and tissue micro-arrays of resected tumors were compared for patients treated at the Leiden University Medical Center (LUMC). When results were discordant, whole-sized slides were analyzed. Additionally, literature was searched for published patient series and combined with our data to assess the concordance of ER and HER2 determination between CNB and resection specimens. RESULTS In the LUMC series, concordance for ER status was 99.1%. Combined concordance from 20 studies and the LUMC patient series was 93.7%. For HER2 testing, concordance was 96.2% for patients in the LUMC series. Our study and three others have investigated the concordance when HER2 was determined according to the American Society of Clinical Oncology and College of Pathology guidelines and overall concordance was 97.8%. CONCLUSIONS Concordance between CNB and surgical specimens was high for both ER and HER2 testing. However, we recommend retesting ER-negative CNB results on the surgical specimen and performing in situ hybridization assays on HER2 immunohistochemistry 3+ CNBs to confirm HER2 status.

Molecularly Defined Adult Granulosa Cell Tumor of the Ovary: The Clinical Phenotype

The histopathologic features of adult granulosa cell tumors (AGCTs) are relatively nonspecific, resulting in misdiagnosis of other cancers as AGCT, a problem that has not been well characterized. FOXL2 mutation testing was used to stratify 336 AGCTs from three European centers into three categories: 1) FOXL2 mutant molecularly defined AGCT (MD-AGCT) (n = 256 of 336), 2) FOXL2 wild-type AGCT (n = 17 of 336), 3) misdiagnosed other tumor types (n = 63 of 336). All statistical tests were two-sided. The overall and disease-specific survival of the misdiagnosed cases was lower than in the MD-AGCTs (P < .001). The misdiagnosed cases accounted for 71.9% of disease-specific deaths within five years. In the population-based cohort, overall survival of MD-AGCT patients was not different from age-matched, population-based controls. Even though 35.2% of all the MD-AGCT patients in our study experienced a relapse, AGCT is usually an indolent disease. The historical, premolecular data underpinning our clinical understanding of AGCT was likely skewed by inclusion of misdiagnosed cases, and future management strategies should reflect the potential for surgical cure and long survival even after relapse.

Discordance in HER2 Status in Gastro-esophageal Adenocarcinomas: A Systematic Review and Meta-analysis

Trastuzumab combined with chemotherapy is standard of care for HER2 positive advanced gastro-esophageal cancers. The reported prevalence of HER2 discordance between primary tumors and corresponding metastases varies, hampering uniform patient selection for HER2 targeted therapy. This meta-analysis explores the influence of HER2 assessment methods on this discordance and investigates the prevalence of HER2 discordance in gastro-esophageal adenocarcinomas. PubMed, Embase and Cochrane databases were searched until January 2016. Differences in discordance rate between strict and broad(er) definitions of HER2 status were assessed using random-effect pair-wise meta-analysis. Random-effect single-arm meta-analyses were performed to assess HER2 discordance and the prevalence of positive and negative conversion. A significantly lower discordance rate in HER2 status between primary tumors and corresponding metastases was observed using a strict vs. broad definition of HER2 status (RR = 0.58, 95%CI 0.41–0.82), with a pooled discordance rate of 6.2% and 12.2%, respectively. Using the strict definition of HER2 assessment pooled overall discordance was 7% (95%CI 5–10%). The lowest discordance rates between primary tumors and corresponding metastasis are observed when using a strict method of HER2 positivity. Treatment outcomes of different studies will be better comparable if selection of eligible patients for HER2 targeted therapy is based on this strict definition.

Direct inhibition of STAT signaling by platinum drugs contributes to their anti-cancer activity

Platinum-based chemotherapeutics are amongst the most powerful anti-cancer drugs. Although their exact mechanism of action is not well understood, it is thought to be mediated through covalent DNA binding. We investigated the effect of platinum-based chemotherapeutics on signaling through signal transducer and activator of transcription (STAT) proteins, which are involved in many oncogenic signaling pathways. We performed in vitro experiments in various cancer cell lines, investigating the effects of platinum chemotherapeutics on STAT phosphorylation and nuclear translocation, the expression of STAT-modulating proteins and downstream signaling pathways. Direct binding of platinum to STAT proteins was assessed using an AlphaScreen assay. Nuclear STAT3 expression was determined by immunohistochemistry and correlated with disease-free survival in retrospective cohorts of head and neck squamous cell carcinoma (HNSCC) patients treated with cisplatin-based chemoradiotherapy (n = 65) or with radiotherapy alone (n = 32). At clinically relevant concentrations, platinum compounds inhibited STAT phosphorylation, resulting in loss of constitutively activated STAT proteins in multiple distinct cancer cell lines. Platinum drugs specifically inhibited phospho-tyrosine binding to SH2 domains, thereby blocking STAT activation, and subsequently downregulating pro-survival- and anti-apoptotic- target genes. Importantly, we found that active STAT3 in tumors directly correlated with response to cisplatin-based chemoradiotherapy in HNSCC patients (p = 0.006). These findings provide insight into a novel, non-DNA-targeted mechanism of action of platinum drugs, and could be leveraged into the use of STAT expression as predictive biomarker for cisplatin chemotherapy and to potentiate other therapeutic strategies such as immunotherapy.Platinum-based chemotherapeutics are amongst the most powerful anti-cancer drugs. Although their exact mechanism of action is not well understood, it is thought to be mediated through covalent DNA binding. We investigated the effect of platinum-based chemotherapeutics on signaling through signal transducer and activator of transcription (STAT) proteins, which are involved in many oncogenic signaling pathways. We performed in vitro experiments in various cancer cell lines, investigating the effects of platinum chemotherapeutics on STAT phosphorylation and nuclear translocation, the expression of STAT-modulating proteins and downstream signaling pathways. Direct binding of platinum to STAT proteins was assessed using an AlphaScreen assay. Nuclear STAT3 expression was determined by immunohistochemistry and correlated with disease-free survival in retrospective cohorts of head and neck squamous cell carcinoma (HNSCC) patients treated with cisplatin-based chemoradiotherapy (n = 65) or with radiotherapy alone (n = 32). At clinically relevant concentrations, platinum compounds inhibited STAT phosphorylation, resulting in loss of constitutively activated STAT proteins in multiple distinct cancer cell lines. Platinum drugs specifically inhibited phospho-tyrosine binding to SH2 domains, thereby blocking STAT activation, and subsequently downregulating pro-survival- and anti-apoptotic- target genes. Importantly, we found that active STAT3 in tumors directly correlated with response to cisplatin-based chemoradiotherapy in HNSCC patients (p = 0.006). These findings provide insight into a novel, non-DNA-targeted mechanism of action of platinum drugs, and could be leveraged into the use of STAT expression as predictive biomarker for cisplatin chemotherapy and to potentiate other therapeutic strategies such as immunotherapy.

The dynamics of HER2 status in esophageal adenocarcinoma

Trastuzumab, a monoclonal antibody against HER2, has become standard of care for metastatic HER2-overexpressing esophagogastric adenocarcinoma and is currently investigated as (neo)adjuvant treatment option in HER2-positive esophagogastric adenocarcinoma. The HER2 status is commonly determined on archived material of the primary tumor. However, this status may change over the course of treatment or disease progression. The aim of this study was to assess the dynamics of HER2 status in esophageal adenocarcinoma (EAC) in patients with resectable and recurrent disease, and to determine the associations of these changes with clinical outcome. Discordance, defined as any change in HER2 status between matched biopsy and post-neoadjuvant chemoradiation therapy resection specimen (N = 170), or between matched resection specimen and recurrence of patients not eligible for curative treatment (N = 61), was determined using the standardized HER2 status scoring system. Clinically relevant positive discordance was defined as a change to HER2 positive status, as this would imply eligibility for HER2-targeted therapy. A difference in HER2 status between biopsy and resection specimen and resection specimen and metachronous recurrence was observed in 2.1% (n = 3) and 3.3% (n = 2) of the paired cases, respectively. Clinically relevant discordance was detected in 1.4% (n = 2) of the resectable patients and 1.6% (n = 1) of the patients with recurrent disease. Patients with HER2-positive status tumors before start of neoadjuvant treatment showed better overall survival, but not statistically significant. No association between HER2 status discordance and survival was found. Clinically relevant HER2 status discordance was observed and in order to prevent under-treatment of patients, the assessment of HER2 status in the metastatic setting should preferably be performed on the most recently developed lesions if the previous HER2 assessment on archival material of the primary tumor was negative.

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.