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// Pieter A. Boonstra 1 , Arja ter Elst 2 , Marco Tibbesma 1, 2 , Lisette J. Bosman 2 , Ron Mathijssen 3 , Florence Atrafi 3 , Frits van Coevorden 4 , Neeltje Steeghs 5 , Sheima Farag 5 , Hans Gelderblom 6 , Winette T.A. van der Graaf 7 , Ingrid M.E. Desar 7 , Jacqueline Maier 8 , Jelle Overbosch 9 , Albert J.H. Suurmeijer 2 , Jourik Gietema 1 , Ed Schuuring 2, * and Anna K.L. Reyners 1, * 1 University of Groningen, University Medical Center Groningen, Department of Medical Oncology, Groningen 9713 GZ, The Netherlands 2 University of Groningen, University Medical Center Groningen, Department of Pathology, Groningen 9713 GZ, The Netherlands 3 Department of Medical Oncology, Erasmus University Medical Center Rotterdam, Rotterdam 3015 CE, The Netherlands 4 Antoni van Leeuwenhoek, Netherlands Cancer Institute, Department of Surgery, Amsterdam 1066 CX, The Netherlands 5 Antoni van Leeuwenhoek, Netherlands Cancer Institute, Department of Medical Oncology, Amsterdam 1066 CX, The Netherlands 6 Leiden University Medical Center, Department of Medical Oncology, Leiden 2300 RC, The Netherlands 7 Radboud University Medical Center, Department of Medical Oncology, Nijmegen 6500 HB, The Netherlands 8 University of Leipzig, Center for Internal Medicine, Department of Hematology/Oncology, Leipzig 04103, Germany 9 University of Groningen, University Medical Center Groningen, Department of Radiology, Groningen 9713 GZ, The Netherlands * These authors share authorship Correspondence to: Anna K.L. Reyners, email: a.k.l.reyners@umcg.nl Keywords: GIST; cell-free circulating DNA; single assay; plasma; digital droplet PCR Received: October 05, 2017 Accepted: January 13, 2018 Published: February 14, 2018 ABSTRACT Background: Gastrointestinal stromal tumors (GISTs) are characterized by oncogenic KIT mutations that cluster in two exon 11 hotspots. The aim of this study was to develop a single, sensitive, quantitative digital droplet PCR (ddPCR) assay for the detection of common exon 11 mutations in both GIST tumor tissue and in circulating tumor DNA (ctDNA) isolated from GIST patients’ plasma. Methods: A ddPCR assay was designed using two probes that cover both hotspots. Available archival FFPE tumor tissue from 27 consecutive patients with known KIT exon 11 mutations and 9 randomly selected patients without exon 11 mutations were tested. Plasma samples were prospectively collected in a multicenter bio-databank from December 2014. ctDNA was analyzed of 22 patients with an exon 11 mutation and a baseline plasma sample. Results: The ddPCR assay detected the exon 11 mutation in 21 of 22 tumors with exon 11 mutations covered by the assay. Mutations in ctDNA were detected at baseline in 13 of 14 metastasized patients, but in only 1 of 8 patients with localized disease. In serial plasma samples from 11 patients with metastasized GIST, a decrease in mutant droplets was detected during treatment. According to RECIST 1.1, 10 patients had radiological treatment response and one patient stable disease. Conclusion: A single ddPCR assay for the detection of multiple exon 11 mutations in ctDNA is a feasible, promising tool for monitoring treatment response in patients with metastasized GIST and should be further evaluated in a larger cohort.

Supplemental Digital Content is available in the text., BRAF mutational testing has become a common practice in the diagnostic process of patients with advanced melanoma. Although time-consuming, DNA sequencing techniques are the current gold standard for mutational testing. However, in certain clinical situations, a rapid test result is required. In this study, the performance of three rapid BRAF mutation tests was compared. Thirty-nine formalin-fixed paraffin-embedded melanoma tissue samples collected between 2007 and 2014 at a single center were included. These samples were analyzed by immunohistochemistry using the anti-BRAF-V600E (VE1) mouse monocolonal antibody (BRAF-VE1 IHC), a V600E-specific Droplet Digital PCR Test, and the Idylla BRAF- Mutation Test (Idylla). Results were compared with the results of conventional BRAF mutation testing, performed using high-resolution melting analysis followed by Sanger sequencing. Next-generation sequencing was performed on samples with discordant results. The Idylla test and Droplet Digital PCR Test correctly identified all mutated and wild-type samples. BRAF-VE1 IHC showed one discordant result. The Idylla test could identify BRAF-V600 mutations other than BRAF-V600E and was the fastest and least laborious test. The Idylla Mutation Test is the most suitable test for rapid BRAF testing in clinical situations on the basis of the broad coverage of treatment-responsive mutations and the fast procedure without the need to perform a DNA isolation step.

Background Gastrointestinal stromal tumors (GISTs) are characterized by oncogenic KIT mutations that cluster in two exon 11 hotspots. The aim of this study was to develop a single, sensitive, quantitative digital droplet PCR (ddPCR) assay for the detection of common exon 11 mutations in both GIST tumor tissue and in circulating tumor DNA (ctDNA) isolated from GIST patients’ plasma. Methods A ddPCR assay was designed using two probes that cover both hotspots. Available archival FFPE tumor tissue from 27 consecutive patients with known KIT exon 11 mutations and 9 randomly selected patients without exon 11 mutations were tested. Plasma samples were prospectively collected in a multicenter bio-databank from December 2014. ctDNA was analyzed of 22 patients with an exon 11 mutation and a baseline plasma sample. Results The ddPCR assay detected the exon 11 mutation in 21 of 22 tumors with exon 11 mutations covered by the assay. Mutations in ctDNA were detected at baseline in 13 of 14 metastasized patients, but in only 1 of 8 patients with localized disease. In serial plasma samples from 11 patients with metastasized GIST, a decrease in mutant droplets was detected during treatment. According to RECIST 1.1, 10 patients had radiological treatment response and one patient stable disease. Conclusion A single ddPCR assay for the detection of multiple exon 11 mutations in ctDNA is a feasible, promising tool for monitoring treatment response in patients with metastasized GIST and should be further evaluated in a larger cohort.

ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using deFuse and EricScript. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing was used to validate fusion genes and digital droplet PCR to validate mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The fusion genes validated in the post-treatment sample were also present in the biopsy before crizotinib. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, fusion genes identified in our study are unlikely to be involved in crizotinib resistance based on presence in pre-treatment biopsies. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance.

Upon transplant, functional HSC clones preferentially expand in certain skeletal locations, exhibiting only limited migration toward other niches., Hematopoietic stem cells (HSCs) are able to migrate through the blood stream and engraft bone marrow (BM) niches. These features are key factors for successful stem cell transplantations that are used in cancer patients and in gene therapy protocols. It is unknown to what extent transplanted HSCs distribute throughout different anatomical niches in the BM and whether this changes with age. Here we determine the degree of hematopoietic migration at a clonal level by transplanting individual young and aged mouse HSCs labeled with barcoded viral vector, followed by assessing the skeletal distribution of hundreds of HSC clones. We detected highly skewed representation of individual clones in different bones at least 11 mo after transplantation. Importantly, a single challenge with the clinically relevant mobilizing agent granulocyte colony-stimulating factor (G-CSF) caused rapid redistribution of HSCs across the skeletal compartments. Old and young HSC clones showed a similar level of migratory behavior. Clonal make-up of blood of secondary recipients recapitulates the barcode composition of HSCs in the bone of origin. These data demonstrate a previously unanticipated high skeletal disequilibrium of the clonal composition of HSC pool long-term after transplantation. Our findings have important implications for experimental and clinical and stem cell transplantation protocols.

Introduction Gastrointestinal stromal tumors (GIST) are rare mesenchymal tumors of the gastrointestinal tract. These tumors are characterized by genomic mutations in the c-KIT gene that drive tumor growth and are target for specific inhibitors. Mutations in exon 11 (single nucleotide variations and deletions) are detected in approximately 70% of GIST. Although unique for each patient, most variations cluster in two different mutational hotspots each 25bp in size. The aim of this study was to develop and test a single quantitative digital droplet PCR (ddPCR) assay to detect exon 11 c-KIT mutations in both formalin fixed paraffin embedded (FFPE) tissue and serial collected cell-free plasma DNA (cfDNA) of GIST patients for diagnostic and treatment response monitoring. Materials and methods The drop-off ddPCR assay for detection of exon 11 mutations consists of two fluorescent (FAM and HEX) labeled probes that each cover one of the 2 mutation hotspots. Since double exon 11 mutations are very rare in GIST, one probe will anneal to the non-mutated sequence (reference probe), while the other probe will not anneal. A decrease of one of the 2 fluorescent signals is considered as the presence of a mutation (“drop-off”). For validation, ddPCR results from tumor biopsies were compared with data from next generation sequencing (NGS) using the IonTorrent platform. Pretreatment FFPE tumor material was obtained from 29 (18 exon 11 mutated, 11 non-exon 11 mutated) GIST patients treated in 2014 and 2015. Plasma samples were collected at baseline (before start of treatment with imatinib) and at consecutive time points during treatment. Results Using the drop-off ddPCR assay on the same DNA used for NGS, mutations in 17 of the 18 samples were detected. Comparison of the allelic fraction of the mutation shows that ddPCR analysis is very similar to NGS. In 11 control samples, who were wild-type (4 samples) or had mutations other than c-KIT exon 11 (7 samples) no loss of signal was detected. CfDNA was analyzed of a 72-year old representative patient with metastatic GIST carrying a c-KIT deletion in exon 11 with a fractional abundance (FA) in tissue of 91%. We detected the mutation using the drop-off ddPCR assay in the plasma sample collected before start of imatinib treatment (FA 16%). Two weeks after initiation of imatinib a rise in mutant allelic frequency was observed (FA 61%) and a decrease after 4 weeks (FA 3%) of therapy. Conclusion The detection of multiple exon 11 mutations/deletions using a single ddPCR assay could be used as a rapid prescreening method for the detection of c-KIT mutations in FFPE tissue in GIST patients. Because of the quantitative nature of this assay, ddPCR analysis might be used to monitor response to treatment. The relevance of this drop-off assay for treatment decision making seems promising and is currently validated on a large series of cfDNA samples and a prospective study in GIST patients (NCT02331914). Citation Format: Pieter A. Boonstra, Marco Tibbesma, Lisette J. Bosman, Ron H.J. Mathijssen, Frits van Coevorden, Neeltje Steeghs, Albert J.H. Suurmeijer, Arja ter Elst, Jourik A. Gietema, Anna K.L. Reyners, Ed M.D. Schuuring, Dutch GIST Consortium. A single ddPCR assay to detect KIT exon 11 mutations in tumor and cell free plasma DNA of patients with gastrointestinal stromal tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3107.