A single digital droplet PCR assay to detect multiple KIT exon 11 mutations in tumor and plasma from patients with gastrointestinal stromal tumors

__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 patien

A single digital droplet PCR assay to detect multipleexon 11 mutations in tumor and plasma from patients with gastrointestinal stromal tumors

Contains fulltext : 190966.pdf (publisher's version ) (Open Access)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

Asymmetry in skeletal distribution of mouse hematopoietic stem cell clones and their equilibration by mobilizing cytokines

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

Genomic Aberrations in Crizotinib Resistant Lung Adenocarcinoma Samples Identified by Transcriptome Sequencing.

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

Summary of the diagnostic FISH, immunohistochemistry and the transcriptome analysis results.

<p>Summary of the diagnostic FISH, immunohistochemistry and the transcriptome analysis results.</p

Patients’ characteristics and fusion products detected in crizotinib resistant tumors.

<p>Patients’ characteristics and fusion products detected in crizotinib resistant tumors.</p

Detection of <i>ALK</i> gene mutations in tumor samples.

<p><b>(A)</b> RNA-sequencing reads of the two mutations. Grey bars show the wild type positions, the colored bar indicates the mutant position. The number of wild type and mutant reads were 56/75 for patient #1 (c.3467G>A) and 0/25 for patient #3 (c.3806G>C)(Top). RNA Sanger sequencing in the post-treatment tumor sample of patient #1 confirmed presence of wild type and mutated <i>EML4-ALK</i> copy using primers covering the <i>ALK</i> break (Bottom). The sequences in this picture are based on plus strand, whereas the <i>ALK</i> gene is located on the minus strand of chromosome 2. <b>(B)</b> DNA Sanger sequencing results in the pre- and post-treatment tumor samples. <b>(C)</b> ddPCR results of the pre-and post-treatment tumor samples of patient #1 and #3. Number of positive droplets for mutant and wild type alleles is written in each gate of the scatter plots. Sensitivity of the assays was 0.1 and 0.5% for C1156Y and G1269A, respectively. Fractional abundance for the mutant allele was 26% and 19.8% in the post-treatment tumors of patients #1 and #3, respectively. Pre: pre-treatment tumor sample; Post: post-treatment tumor sample.</p