Pepijn Schellen

RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics

Summary Tumor-educated blood platelets (TEPs) are implicated as central players in the systemic and local responses to tumor growth, thereby altering their RNA profile. We determined the diagnostic potential of TEPs by mRNA sequencing of 283 platelet samples. We distinguished 228 patients with localized and metastasized tumors from 55 healthy individuals with 96% accuracy. Across six different tumor types, the location of the primary tumor was correctly identified with 71% accuracy. Also, MET or HER2-positive, and mutant KRAS, EGFR, or PIK3CA tumors were accurately distinguished using surrogate TEP mRNA profiles. Our results indicate that blood platelets provide a valuable platform for pan-cancer, multiclass cancer, and companion diagnostics, possibly enabling clinical advances in blood-based “liquid biopsies”.

Rearranged EML4-ALK fusion transcripts sequester in circulating blood platelets and enable blood-based crizotinib response monitoring in non-small-cell lung cancer

Purpose: Non-small-cell lung cancers harboring EML4-ALK rearrangements are sensitive to crizotinib. However, despite initial response, most patients will eventually relapse, and monitoring EML4-ALK rearrangements over the course of treatment may help identify these patients. However, challenges associated with serial tumor biopsies have highlighted the need for blood-based assays for the monitoring of biomarkers. Platelets can sequester RNA released by tumor cells and are thus an attractive source for the non-invasive assessment of biomarkers. Methods: EML4-ALK rearrangements were analyzed by RT-PCR in platelets and plasma isolated from blood obtained from 77 patients with non-small-cell lung cancer, 38 of whom had EML4-ALK-rearranged tumors. In a subset of 29 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, EML4-ALK rearrangements in platelets were correlated with progression-free and overall survival. Results: RT-PCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets. In the subset of 29 patients treated with crizotinib, progression-free survival was 3.7 months for patients with EML4-ALK+ platelets and 16 months for those with EML4-ALK− platelets (hazard ratio, 3.5; P = 0.02). Monitoring of EML4-ALK rearrangements in the platelets of one patient over a period of 30 months revealed crizotinib resistance two months prior to radiographic disease progression. Conclusions: Platelets are a valuable source for the non-invasive detection of EML4-ALK rearrangements and may prove useful for predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based on radiographic imaging alone.

WEE1 Kinase Inhibition Enhances the Radiation Response of Diffuse Intrinsic Pontine Gliomas

Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric disease. Thus far, no therapeutic agent has proven beneficial in the treatment of this malignancy. Therefore, conventional DNA-damaging radiotherapy remains the standard treatment, providing transient neurologic improvement without improving the probability of overall survival. During radiotherapy, WEE1 kinase controls the G2 cell-cycle checkpoint, allowing for repair of irradiation (IR)-induced DNA damage. Here, we show that WEE1 kinase is one of the highest overexpressed kinases in primary DIPG tissues compared with matching non-neoplastic brain tissues. Inhibition of WEE1 by MK-1775 treatment of DIPG cells inhibited the IR-induced WEE1-mediated phosphorylation of CDC2, resulting in reduced G2–M arrest and decreased cell viability. Finally, we show that MK-1775 enhances the radiation response of E98-Fluc-mCherry DIPG mouse xenografts. Altogether, these results show that inhibition of WEE1 kinase in conjunction with radiotherapy holds potential as a therapeutic approach for the treatment of DIPG. Mol Cancer Ther; 12(2); 141–50. ©2012 AACR.

Subventricular spread of diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is the second most common malignant pediatric brain tumor and the leading cause of brain tumor death in childhood [1]. 80 % of DIPG tumors exhibit a specific mutation (H3K27M) in the genes encoding histone 3.1 or 3.3 [2, 3]. standard therapy consisting of local radiotherapy to a dosage of 54–60 Gy extends median survival from 5 months to ∼9 months; 5-year survival remains less than 1 % [1]. The practice of focal radio-therapy to the brainstem is based in part on a 1982 autopsy study reporting DIPG to be relatively localized to the pons and adjacent structures [4]. In contrast, other neuroimaging and autopsy studies have identified widespread disease including supratentorial extension and leptomeningeal spread [5, 6]. Here, we report an autopsy series of 16 patients evaluated from 2009–2014 at stanford (n = 10) and VU (n = 6) University Medical Centers [7]. patient characteristics are listed in Table S1. Consistent with previous reports [5, 6], we found widespread dissemination of DIPG with extension to midbrain and medulla in 63 %, cerebellum in 56 %, thalamus in 56 %, frontal cortex in 25 % and supratentorial leptomeninges in 25 % (Fig. 1). The spinal cord was not consistently examined, but metastases were found in two of three cases examined; both had clinical evidence of spinal cord spread. Fig. 1 Extent of spread in DIPG. a Neuroanatomical sites and frequency of tumor invasion. Numbers indicate the percentage of cases that exhibit tumor invasion at the indicated anatomical location. The size of the circles marking each anatomical site (color key ... A previously under-recognized pattern of subventricular spread was noted in 10/16 cases, with infiltration of the subventricular zone (SVZ) and tumor nodules in the frontal horns of the lateral ventricles. In three cases lateral ventricular disease was noted on pre-mortem MRI (Fig. 2a), but subclinical tumor invasion in the SVZ of the lateral ventricles was found in six additional cases; subventricular spread was found in the third ventricle of one additional case (Fig. 2). The observed pattern of ventricular/subventricular involvement could be due to direct invasion along the SVZ corridor, intraventricular cerebrospinal fluid (CSF) seeding of the SVZ, or an as yet undescribed mechanism. The postnatal SVZ is a neural stem cell niche in the human brain [8] and DIPG cells express an immunophenotype reminiscent of neural precursor cells (Fig. S1 and [9]). Whether DIPG cells exhibit a particular tropism for this niche remains to be explored. Fig. 2 Invasion of the subventricular zone in DIPG (a). MRI images illustrating enhancing lesions (T1 post gadolinium, left image) at the frontal horns of the lateral ventricles with associated edema (FLAIR, right image) in case SU-DIPG-XIII. b H&E ( ... Following standard brainstem radiotherapy, disease progression typically occurs locally in the brainstem. However, in three of sixteen cases the subventricular frontal lobe disease contributed substantially to morbidity and mortality and preceded pontine recurrence in two cases. As therapies improve and patients survive longer in the natural history of their cancer, new patterns of regional relapse often appear (e.g. sanctuary disease in childhood leukemia). Our data show subventricular tumor spread in the majority of patients, typically later in the course of their disease. Thus as future therapies evolve to control local disease, strategies including extended or whole brain irradiation may become crucial. The patterns of widespread dissemination, including leptomeningeal, direct extension and subventricular spread, suggest that the extent of the optimal radiation field should be re-examined.

Swarm Intelligence-Enhanced Detection of Non-Small-Cell Lung Cancer Using Tumor-Educated Platelets

Summary Blood-based liquid biopsies, including tumor-educated blood platelets (TEPs), have emerged as promising biomarker sources for non-invasive detection of cancer. Here we demonstrate that particle-swarm optimization (PSO)-enhanced algorithms enable efficient selection of RNA biomarker panels from platelet RNA-sequencing libraries (n = 779). This resulted in accurate TEP-based detection of early- and late-stage non-small-cell lung cancer (n = 518 late-stage validation cohort, accuracy, 88%; AUC, 0.94; 95% CI, 0.92–0.96; p < 0.001; n = 106 early-stage validation cohort, accuracy, 81%; AUC, 0.89; 95% CI, 0.83–0.95; p < 0.001), independent of age of the individuals, smoking habits, whole-blood storage time, and various inflammatory conditions. PSO enabled selection of gene panels to diagnose cancer from TEPs, suggesting that swarm intelligence may also benefit the optimization of diagnostics readout of other liquid biopsy biosources.

Convection enhanced delivery of carmustine to the murine brainstem: a feasibility study.

BACKGROUND Systemic delivery of therapeutic agents remains ineffective against diffuse intrinsic pontine glioma (DIPG), possibly due to an intact blood-brain-barrier (BBB) and to dose-limiting toxicity of systemic chemotherapeutic agents. Convection-enhanced delivery (CED) into the brainstem may provide an effective local delivery alternative for DIPG patients. NEW METHOD The aim of this study is to develop a method to perform CED into the murine brainstem and to test this method using the chemotherapeutic agent carmustine (BiCNU). To this end, a newly designed murine CED catheter was tested in vitro and in vivo. After determination of safety and distribution, mice bearing VUMC-DIPG-3 and E98FM-DIPG brainstem tumors were treated with carmustine dissolved in DW 5% or carmustine dissolved in 10% ethanol. RESULTS Our results show that CED into the murine brainstem is feasible and well tolerated by mice with and without brainstem tumors. CED of carmustine dissolved in 5% DW increased median survival of mice with VUMC-DIPG-3 and E98FM-DIPG tumors with 35% and 25% respectively. Dissolving carmustine in 10% ethanol further improved survival to 45% in mice with E98FM-DIPG tumors. COMPARISON WITH EXISTING METHODS Since genetically engineered and primary DIPG models are currently only available in mice, murine CED studies have clear advantages over CED studies in other animals. CONCLUSION CED in the murine brainstem can be performed safely, is well tolerated and can be used to study efficacy of chemotherapeutic agents orthotopically. These results set the foundation for more CED studies in murine DIPG models.

Deceptive morphologic and epigenetic heterogeneity in diffuse intrinsic pontine glioma

Historically, the diagnosis of diffuse intrinsic pontine glioma (DIPG) was based on typical imaging findings and clinical characteristics instead of pathology. However, the discovery of mutations in histone H3 variants, and the availability of tumor material for molecular analysis, has led to a paradigm shift in DIPG research and clinical practice. Using data from whole-brain autopsies in a series of nine DIPG patients with known histone mutational status, we here aim to review histopathological characteristics with special focus on intratumoral heterogeneity (ITH) and histone 3 K27 trimethylation (H3 K27me3). All DIPGs showed marked histologic ITH, with 56% even showing focal areas resembling a WHO grade I phenotype. As expected, H3 K27me3 immunoreactivity was lost in the tumors that were H3 K27M-mutated (seven patients; 67% H3.3, 11% H3.1). Strikingly, the H3K27 wildtype tumors (two patients; 22%) also contained H3 K27me3-immunonegative areas. Our study underscores the importance of the choice of the biopsy site, as ITH in DIPGs could theoretically lead to erroneous histological diagnoses with small biopsies. New in this respect is our finding that a substantial number of otherwise typical DIPGs has areas resembling WHO grade I tumors (esp. pilocytic astrocytoma, subependymoma). Furthermore, our study shows that negative H3 K27me3 immunohistochemistry in a DIPG does not imply a H3 K27-mutant tumor.

Structural Alterations of MET Trigger Response to MET Kinase Inhibition in Lung Adenocarcinoma Patients

Purpose: We sought to investigate the clinical response to MET inhibition in patients diagnosed with structural MET alterations and to characterize their functional relevance in cellular models. Experimental Design: Patients were selected for treatment with crizotinib upon results of hybrid capture–based next-generation sequencing. To confirm the clinical observations, we analyzed cellular models that express these MET kinase alterations. Results: Three individual patients were identified to harbor alterations within the MET receptor. Two patients showed genomic rearrangements, leading to a gene fusion of KIF5B or STARD3NL and MET. One patient diagnosed with an EML4-ALK rearrangement developed a MET kinase domain duplication as a resistance mechanism to ceritinib. All 3 patients showed a partial response to crizotinib that effectively inhibits MET and ALK among other kinases. The results were further confirmed using orthogonal cellular models. Conclusions: Crizotinib leads to a clinical response in patients with MET rearrangements. Our functional analyses together with the clinical data suggest that these structural alterations may represent actionable targets in lung cancer patients. Clin Cancer Res; 24(6); 1337–43. ©2017 AACR.