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Structural variants (SVs) caused by chromosomal rearrangements in common fragile sites or long interspersed nuclear element (LINE) retrotranspositions are highly prevalent in colorectal cancer. However, methodology for the targeted detection of these SVs is lacking. This article reports the use of formalin-fixed paraffin-embedded targeted-locus capture (FFPE-TLC) sequencing as a novel technology for the targeted detection of tumor-specific SVs. Analysis of 29 FFPE colorectal tumor samples and 8 matched normal samples revealed tumor-specific SVs in 24 patients (83%), with a median of 2 SVs per patient (range, 1 to 21). A total of 104 SVs were found in the common fragile site-associated genes MACROD2, PRKN, FHIT, and WWOX in 18 patients (62%), and 39 SVs caused by three LINE transposable elements were found in 15 patients (52%). Tumor specificity of SVs was independently verified by Droplet Digital PCR of tumor tissue DNA, and their applicability as plasma circulating tumor DNA biomarkers was demonstrated. It was concluded that FFPE-TLC sequencing enables the detection of tumor-specific SVs caused by chromosomal rearrangements and LINE retrotranspositions in FFPE tissue. Therefore, FFPE-TLC sequencing facilitates the investigation of the biological and clinical effects of SVs using FFPE material from (retrospective) cohorts of cancer patients and has potential clinical applicability in the detection of SV biomarkers in the routine molecular diagnostics setting., Competing Interests: Disclosure Statement C.R.-A. declares nonfinancial support from Personal Genome Diagnostics and Cergentis BV. E.S., J.S., and H.F. are employees of Cergentis BV (a Solvias company), the inventor and owner of the patents on FFPE-TLC technology. S.L. reports nonfinancial support from Cergentis BV and a patent pending. E.J.v.B. reports nonfinancial support from Cergentis BV. D.v.d.B. has provided lectures, expert testimony, and advisory board presence to Roche Diagnostics, all outside the submitted work and all financial supports transferred to institute. G.A.M. is co-founder and board member (CSO) of CRCbioscreen BV; has had research collaboration with CZ Health Insurances (cash matching to ZonMW grant); has had research collaborations with Exact Sciences, Sysmex, Sentinel Ch SpA, Personal Genome Diagnostics, DELFi, and HMF (these companies provide materials, equipment and/or sample/genomic analyses); and has several patents pending/issued. S.A. reports public–private partnership consortia grants in collaboration with Cergentis BV, Olink Proteomics AB, and Quanterix Corporation and has a patent pending. R.J.A.F. reports public–private partnership consortia grants in collaboration with Cergentis BV; reports public–private partnership consortia grants and nonfinancial support in collaboration with Personal Genome Diagnostics, Delfi, Natera, and Merck BV outside the submitted work; and has several patents pending., (Copyright © 2024. Published by Elsevier Inc.)

Human papillomavirus (HPV) infections are an increasing cause of oropharyngeal squamous cell carcinomas (OPSCC). Integration of the viral genome into the host genome is suggested to affect carcinogenesis, however, the correlation with OPSCC patient prognosis is still unclear. Research on HPV integration is hampered by current integration detection technologies and their unsuitability for formalin-fixed paraffin-embedded (FFPE) tissues. This study aims to develop and validate a novel targeted proximity-ligation based sequencing method (targeted locus amplification/capture [TLA/TLC]) for HPV integration detection in cell lines and FFPE OPSCCs. For the identification of HPV integrations, TLA/TLC was applied to 7 cell lines and 27 FFPE OPSCCs. Following preprocessing steps, a polymerase chain reaction (PCR)-based HPV enrichment was performed on the cell lines and a capture-based HPV enrichment was performed on the FFPE tissues before paired-end sequencing. TLA was able to sequence up to hundreds of kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with detection of integrated papillomavirus sequences PCR data and the literature. TLC detected integrated HPV in 15/27 FFPE OPSCCs and identified simple and complex integration patterns. In general, TLA/TLC confirmed PCR data and detected additional integration sites. In conclusion TLA/TLC reliably and robustly detects HPV integration in cell lines and FFPE OPSCCs, enabling large, population-based studies on the clinical relevance of HPV integration. Furthermore, this approach might be valuable for clonality assessment of HPV-related tumors in clinical diagnostics., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Background: Liquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors., Materials and Methods: Regions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed., Results: TLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse., Conclusion: We demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols., Competing Interests: IS, ESt, JSw, HF, MM, ESp are employees of Cergentis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2023 van Zogchel, Lak, Gelineau, Sergeeva, Stelloo, Swennenhuis, Feitsma, van Min, Splinter, Bleijs, Groot Koerkamp, Breunis, Meister, Kholossy, Holstege, Molenaar, de Leng, Stutterheim, van der Schoot and Tytgat.)

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.

Here we present the Puncher technology for the isolation of single cells. This technology combines a silicon chip with microwells, fluorescence imaging, and a punching method to isolate and transfer the single cells to standard reaction tubes. The technology is compatible with commercially available downstream workflows and instrumentation. Here we focus on the isolation of CTC but the Puncher technology can be applied to isolate single cells from liquid biopsies and more general from cell suspensions. It is especially suited for cell suspensions that contain: Cells of interest at a frequency of 1 per 10,000 or less A low total number of cells ranging from 1 to 100,000, that are present in a volume of 0.01 to 50 mL. The frequency of appearance of CTC in blood is in the order of the 1 per 10 6 leukocytes. To be able to isolate the single CTC with the Puncher technology, enrichment of the CTC by a 3 logs reduction of the leukocytes is required. Here we describe the use of Rosettesep and Parsortix as examples of pre-enrichment methods that are compatible with the Puncher technology and further downstream applications. © 2018 International Society for Advancement of Cytometry., (© 2018 International Society for Advancement of Cytometry.)

Purpose: Circulating tumor cells (CTCs) have clinical relevance, but their study has been limited by their low frequency. Experimental Design: We evaluated liquid biopsies by apheresis to increase CTC yield from patients suffering from metastatic prostate cancer, allow precise gene copy-number calls, and study disease heterogeneity. Results: Apheresis was well tolerated and allowed the separation of large numbers of CTCs; the average CTC yield from 7.5 mL of peripheral blood was 167 CTCs, whereas the average CTC yield per apheresis (mean volume: 59.5 mL) was 12,546 CTCs. Purified single CTCs could be isolated from apheresis product by FACS sorting; copy-number aberration (CNA) profiles of 185 single CTCs from 14 patients revealed the genomic landscape of lethal prostate cancer and identified complex intrapatient, intercell, genomic heterogeneity missed on bulk biopsy analyses. Conclusions: Apheresis facilitated the capture of large numbers of CTCs noninvasively with minimal morbidity and allowed the deconvolution of intrapatient heterogeneity and clonal evolution. Clin Cancer Res; 24(22); 5635-44. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Introduction: The CellSearch® CTC test enumerates tumor cells present in 7.5 ml blood of cancer patients. improvements, extensions and different utilities of the cellsearch system are discussed in this paper. Areas covered: This paper describes work performed with the CellSearch system, which go beyond the normal scope of the test. All results from searches with the search term 'CellSearch' from Web of Science and PubMed were categorized and discussed. Expert commentary: The CellSearch Circulating Tumor Cell test captures and identifies tumor cells in blood that are associated with poor clinical outcome. How to best use CTC in clinical practice is being explored in many clinical trials. The ability to extract information from the CTC to guide therapy will expand the potential clinical utility of CTC.

We present a novel electrode configuration consisting of coplanar side electrode pairs integrated at the half height of the microchannels for the creation of a homogeneous electric field distribution as well as for synchronous optical and electrical measurements. For the integration of such electrodes in fused silica microsystems, a dedicated microfabrication method was utilized, whereby an intermediate bonding layer was applied to lower the temperature for fusion bonding to avoid thereby metal degradation and subsequently to preserve the electrode structures. Finally, we demonstrate the applicability of our devices with integrated electrodes for single cell electrical lysis and simultaneous fluorescence and impedance measurements for both cell counting and characterization.

Background: The objective of this study was to detect and quantify circulating tumour cells (CTC) in peripheral and portal blood of patients who had open or laparoscopic surgery for primary colonic cancer., Methods: Patients in the laparoscopic-group were operated on in a medial to lateral approach ("vessels first"), in the open-group a lateral to medial approach was applied. The enumeration of CTC was performed with the CellSearch System. Intra-operative samples were taken paired-wise (from peripheral and portal circulation) directly after entering the abdominal cavity (T1), after mobilisation of the tumour baring segment (T2), and after tumour resection (T3). Ploidy of both the CTC and tissue of the primary tumour was determined for chromosome 1, 7, 8 and 17., Results: Thirty-one patients were included; 18 patients had open surgery, 13 patients were operated on laparoscopically. The percentage of samples with CTC at T1 was 7% in peripheral blood and 54% in portal blood (p=0.002). At T2, 4% and 31% respectively (p=0.031). And at T3, 4% and 26% respectively (p=0.125). The cumulative percentage of samples with CTC was significantly higher during open surgery as compared to the laparoscopic approach. Both the CTC and tissue of the primary tumour were diploid for chromosome 1, 7, 8 and 17., Conclusion: The detection rate and quantity of CTC is significantly increased intra-operatively and is significantly higher in portal blood compared to peripheral blood. Significantly less CTC were detected during laparoscopic surgery probably as result of the medial to lateral approach.

It has now been well documented that the normal and tumorous canine mammary glands can be extra-pituitary sites of substantial growth hormone (GH) synthesis. Until now, attempts to reproduce the GH synthesis in-vitro using canine mammary explants or mammary tumor cells have not been successful. Therefore, the response of CMT-U335 canine mammary tumor cells to administered porcine GH (pGH) was investigated as an in-vitro model to study the possible effects of GH synthesis on this site. CMT-U335 cells spontaneously express the growth hormone receptor (GHR) as well as the prolactin receptor (PRLR). Twenty five minutes after administration, GH induced, in a dose-dependent manner, phosphorylation of the transcription factors Stat5a and Stat5b. Clear phosphorylation was induced by 10(-7) M and 10(-8) M pGH, with virtually no phosphorylation at 10(-9) M pGH. A similar dose-dependent phosphorylation of Stat5a by ovine prolactin was found in these cells. Although at high concentrations binding of pGH to the canine PRLR can occur (albeit with a low pKa), the similar dose-dependent effect of oPRL on Stat5a phosphorylation indicated that pGH signaled through the GHR. Remarkably, pGH induced a moderately decreased proliferation of CMT-U335 tumor cells, which may indicate that GH induces differentiation in these tumor cells. The GH-induced activation of Stat5a and Stat5b in these cells, as part of the JAK/Stat signal transduction pathway, is consistent with mammary GH playing a role in autocrine and/or paracrine stimulation of (tumorous) mammary cells.

GH synthesis has been documented in canine mammary tissue and mammary tumors. In the present report, the characteristics of the GH receptor (GHR) are studied in these tissues. First, using immunohistochemistry, GHR was found to be present throughout normal and tumorous mammary tissues, being localized in epithelial and myoepithelial/spindle cell components and in the activated fibroblasts of desmoplastic tumor stroma. GHR expression seemed to be down-regulated only in terminally differentiated alveoli in normal tissue. GHR immunoreactivity in particular mammary (adeno)carcinomas was heterogenous. Second, the canine GHR was characterized at the molecular level. Northern blot analysis revealed a major GHR transcript of approximately 4.2 kb. The coding sequence of the canine GHR shows extensive homology with the GHR of several species. Seminested RT-PCR (using primers annealing in exons 4-5, exon 6, and exon 9) generated, next to the primary product, four different products in mammary tissues and the canine mammary tumor cell line CMT-U335, which seemed to be alternative GHR transcripts. These alternative GHR transcripts were characterized by exon 8 skipping, exon 7 skipping, and use of alternative splice donor and acceptor sites. Especially, the transcript that is missing exon 8 may encode a GH binding protein. In most malignant mammary samples, only the primary transcript was present; and alternative transcripts could not be detected. The absence of alternative GHR transcripts in mammary carcinomas, and thus putative inhibitors of GH-induced signal transduction, may contribute to enhanced sensitivity of malignant tumors to GH.