Construction of repeat-free fluorescence in situ hybridization probes

FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C0t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes

Analysis of Released Circulating Tumor Cells During Surgery for Non-Small Cell Lung Cancer:are they what they appear to be?

Purpose: Tumor cells from patients with lung cancer are expelled from the primary tumor into the blood, but difficult to detect in the peripheral circulation. We studied the release of circulating tumor cells (CTCs) during surgery to test the hypothesis that CTC counts are influenced by hemodynamic changes (caused by surgical approach) and manipulation. Experimental Design: Patients undergoing video-assisted thoracic surgery (VATS) or open surgery for (suspected) primary lung cancer were included. Blood samples were taken before surgery (T0) from the radial artery (RA), from both the RA and pulmonary vein (PV) when the PV was located (T1) and when either the pulmonary artery (T2 open) or the PV (T2VATS) was dissected. The CTCs were enumerated using the CellSearch system. Single-cell whole-genome sequencing was performed on isolated CTCs for aneuploidy. Results: CTCs were detected in 58 of 138 samples (42%) of 31 patients. CTCs were more often detected in the PV (70%) compared with the RA (22%, P <0.01) and in higher counts ( P <0.01). After surgery, the RA but not the PV showed less often CTCs (P = 0.02). Type of surgery did not influence CTC release. Only six of 496 isolated CTCs showed aneuploidy, despite matched primary tumor tissue being aneuploid. Euploid so-called CTCs had a different morphology than aneuploid. Conclusions: CTCs defined by CellSearch were identified more often and in higher numbers in the PV compared with the RA, suggesting central clearance. The majority of cells in the PV were normal epithelial cells and outnumbered CTCs. Release of CTCs was not influenced by surgical approach

Formalin-Fixed, Paraffin-Embedded–Targeted Locus Capture:A Next-Generation Sequencing Technology for Accurate DNA-Based Gene Fusion Detection in Bone and Soft Tissue Tumors

Chromosomal rearrangements are important drivers in cancer, and their robust detection is essential for diagnosis, prognosis, and treatment selection, particularly for bone and soft tissue tumors. Current diagnostic methods are hindered by limitations, including difficulties with multiplexing targets and poor quality of RNA. A novel targeted DNA-based next-generation sequencing method, formalin-fixed, paraffin-embedded–targeted locus capture (FFPE-TLC), has shown advantages over current diagnostic methods when applied on FFPE lymphomas, including the ability to detect novel rearrangements. We evaluated the utility of FFPE-TLC in bone and soft tissue tumor diagnostics. FFPE-TLC sequencing was successfully applied on noncalcified and decalcified FFPE samples (n = 44) and control samples (n = 19). In total, 58 rearrangements were identified in 40 FFPE tumor samples, including three previously negative samples, and none was identified in the FFPE control samples. In all five discordant cases, FFPE-TLC could identify gene fusions where other methods had failed due to either detection limits or poor sample quality. FFPE-TLC achieved a high specificity and sensitivity (no false positives and negatives). These results indicate that FFPE-TLC is applicable in cancer diagnostics to simultaneously analyze many genes for their involvement in gene fusions. Similar to the observation in lymphomas, FFPE-TLC is a good DNA-based alternative to the conventional methods for detection of rearrangements in bone and soft tissue tumors.</p

Single tube liquid biopsy for advanced non-small cell lung cancer

The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing. We studied the relation between overall survival (OS) and the presence of four cancer biomarkers from a single blood draw in advanced NSCLC patients: EpCAM(high) circulating tumor cells (CTC), EpCAM(low) CTC, tumor-derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA). EpCAM(high) CTC were detected with CellSearch, tdEV in the CellSearch images and EpCAM(low) CTC with filtration after CellSearch. ctDNA was isolated from plasma and mutations present in the primary tumor were tracked with deep sequencing methods. In 97 patients, 21% had >= 2 EpCAM(high) CTC, 15% had >= 2 EpCAM(low) CTC, 27% had >= 18 tdEV and 19% had ctDNA with >= 10% mutant allele frequency. Either one of these four biomarkers could be detected in 45% of the patients and all biomarkers were present in 2%. In 11 out of 16 patients (69%) mutations were detected in the ctDNA. Two or more unfavorable biomarkers were associated with poor OS. The presence of EpCAM(high) CTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAM(low) CTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy

Development of automatic FISH probe counting in CTC

Introduction: Presence of Circulating Tumor Cells (CTC) in blood of patients with metastatic carcinomas has been associated with poor progression free and overall survival. Characterization of CTC can be performed by Fluorescence In Situ Hybridization (FISH), however counting of FISH dots by human reviewers can be tiring and subjective and thus likely produces variable outcomes. We investigated whether automated counting of FISH dots in CTC is comparable to the counts obtained by expert reviewers. Material and Methods: Samples processed on the CellSearchTM system for CTC counting were hybridized with fluorescent DNA probes targeting the HER2/neu gene region and the centromeric region of chromosome 17 or the centromeric regions of chromosome 1,7,8,17. (1) For optimization of the algorithm 492 Z-stacks from leukocytes carried over through the CellSearch procedure were recorded and a maximum intensity profile (MIP) was created. Five reviewers counted FISH dots in the MIP data set to create a ground truth. The automatic counting algorithm was validated in a set of stored images of CTC probed for chromosome 1,7,8,17 from castration resistant prostate cancer patients (CRPC).(1) Results: The data set with carried-over leukocytes was counted reliably by the algorithm: 97.8% of the HER2/neu FISH dots and 97.5% of the centromeric 17 dots were counted equally by the PC and the reviewers, regarding only the subset of images for which all the reviewers agreed. The mean intra-reviewer agreement was 96.5%. In the validation set copy number of chromosome 1 in carried-over leukocytes scored by an expert agreed in 50.8% with the automated count, for chromosome 7 34.4% for chromosome 8 22.8.% and for chromosome 17 55.0%. For CTC in the validation set copy number of chromosome 1 scored by an expert agreed in 71.6% with the automated count, for chromosome 7 56.2% for chromosome 8 64.8% and for chromosome 17 41.0%. For copy numbers larger than 6 both the expert and automated count were recorded as larger than 6. Agreement between the expert count and automated count did not significantly alter with the detected copy number. Over-count in the validation set was largely due to clustering of nuclei that were counted as one cell. Conclusions: Automatic FISH dot counting in CTC images is feasible for images where reviewers agree upon. The intra-reviewer variation of 3.5% shows that reviewers have ambiguous rules and are not reliable. This variation is closely related to the heterogeneity -size and shape- of the FISH dots. The PC has a reproducibility of 100% and is thus a good replacement for human reviewers

Parallel single cancer cell whole genome amplification using button-valve assisted mixing in nanoliter chambers

The heterogeneity of tumor cells and their alteration during the course of the disease urges the need for real time characterization of individual tumor cells to improve the assessment of treatment options. New generations of therapies are frequently associated with specific genetic alterations driving the need to determine the genetic makeup of tumor cells. Here, we present a microfluidic device for parallel single cell whole genome amplification (pscWGA) to obtain enough copies of a single cell genome to probe for the presence of treatment targets and the frequency of its occurrence among the tumor cells. Individual cells were first captured and loaded into eight parallel amplification units. Next, cells were lysed on a chip and their DNA amplified through successive introduction of dedicated reagents while mixing actively with the help of integrated button-valves. The reaction chamber volume for scWGA 23.85 nl, and starting from 6-7 pg DNA contained in a single cell, around 8 ng of DNA was obtained after WGA, representing over 1000-fold amplification. The amplified products from individual breast cancer cells were collected from the device to either directly investigate the amplification of specific genes by qPCR or for re-amplification of the DNA to obtain sufficient material for whole genome sequencing. Our pscWGA device provides sufficient DNA from individual cells for their genetic characterization, and will undoubtedly allow for automated sample preparation for single cancer cell genomic characterization

Characterization of circulating tumor cells by fluorescence in situ hybridization

Tumor cells in blood of patients with metastatic carcinomas have been associated with poor survival prospects. Further characterization of these cells may provide further insights into the metastatic process. Circulating Tumor Cells (CTC) were enumerated in 7.5 mL of blood with the CellSearch™ system. After enumeration of Cytokeratin+, CD45−, nucleated cells, the cells are fixed in the cartridge while maintaining their original position. Cartridges were hybridized with FISH probes against the centromeric regions of chromosome 1, 7, 8, and 17. Next fluorescence images of the FISH probes of the previous identified CTC were acquired. Leukocytes surrounding the CTC were used as internal controls. The number of copies of chromosome 1, 7, 8, and 17 could be determined in 118 CTC containing blood samples from 59 metastatic prostate cancer patients. The samples contained a total of 21,751 CTC (mean 184, median 16, SD 650). Chromosome counts were obtained in 61% of the relocated CTC. On an average, these CTC contained 2.8 copies of chromosome 1, 2.7 copies of chromosome 7, 3.1 copies of chromosome 8, and 2.3 copies of chromosome 17. CTC in which no chromosome count was obtained most likely underwent apoptosis indicated by the expression of M30. In 6/59 patients only diploid CTC were detected these samples, however, only contained 1–5 CTC. Heterogeneity in the chromosomal abnormalities was observed between CTC of different patients as well as among CTC of the same patient. Cytogenetic composition of CTC can be reliably assessed after they have been identified by the CellSearch™ system. The majority of CTC in hormone refractory prostate cancer are aneuploid confirming that they indeed are cancer cells. An extensive heterogeneity in the copy number of each of the chromosomes was observed

Big city life in the Second Commonwealth of Poland in Polish interwar feature films. An Introduction

Polish feature films from the interwar period are to a great extent a reflection of the reality of that time. This can be seen mainly in the presentation of particular architectural objects and fashion from that time. The films are also a source of knowledge about the problems of the time: the impoverishment of the society, economic crisis, prostitution, etc. These pictures reflect reality, yet they are subject to numerous transformations, as the result of the conventions of the chosen genre.Polski film fabularny omawianego okresu jest w znaczącym stopniu odbiciem ówczesnej rzeczywistości. Objawia się to głównie w dziedzinie urbanistyki, czyli przedstawiania na ekranie określonych obiektów architektonicznych, oraz w dziedzinie panującej wówczas mody. Jest ponadto źródłem wiedzy na temat typowych dla tego okresu problemów społecznych (pauperyzacja społeczeństwa, kryzys gospodarczy, prostytucja etc.). Obrazy te posiadają z jednej strony znamiona prawdy ekranowej, z drugiej – podane są licznym przekształceniom wynikającym z przyjętej konwencji gatunkowej