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Background Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient–derived plasma samples. Methods Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. Results Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane–based methods, samples extracted with magnetic beads–based kits revealed an overall lower total ccfDNA yield (−29%; P Conclusions In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane–based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies.

IntroductionThe locoregional failure (LRF) rate in human papilloma virus (HPV)-negative oropharyngeal squamous cell carcinoma (OPSCC) remains disappointingly high and toxicity is substantial. Response prediction prior to or early during treatment would provide opportunities for personalised treatment. Currently, there are no accurate predictive models available for correct OPSCC patient selection. Apparently, the pivotal driving forces that determine how a OPSCC responds to treatment, have yet to be elucidated. Therefore, the holistiC early respOnse assessMent for oroPharyngeaL cancer paTiEnts study focuses on a holistic approach to gain insight in novel potential prognostic biomarkers, acquired before and early during treatment, to predict response to treatment in HPV-negative patients with OPSCC.Methods and analysisThis single-centre prospective observational study investigates 60 HPV-negative patients with OPSCC scheduled for primary radiotherapy (RT) with cisplatin or cetuximab, according to current clinical practice. A holistic approach will be used that aims to map the macroscopic (with Intra Voxel Incoherent Motion Diffusion Kurtosis Imaging (IVIM-DKI); before, during, and 3 months after RT), microscopic (with biopsies of the primary tumour acquired before treatment and irradiated ex vivo to assess radiosensitivity), and molecular landscape (with circulating tumour DNA (ctDNA) analysed before, during and 3 months after treatment). The main end point is locoregional control (LRC) 2 years after treatment. The primary objective is to determine whether a relative change in the mean of the diffusion coefficient D (an IVIM-DKI parameter) in the primary tumour early during treatment, improves the performance of a predictive model consisting of tumour volume only, for 2 years LRC after treatment. The secondary objectives investigate the potential of other IVIM-DKI parameters, ex vivo sensitivity characteristics, ctDNA, and combinations thereof as potential novel prognostic markers.Ethics and disseminationThe study was approved by the Medical Ethical Committee of Erasmus Medical Center. The main results of the trial will be presented in international meetings and medical journals.Trial registration numberNL8458.

Quantification of tumor-specific variants (TSVs) in cell-free DNA is rapidly evolving as a prognostic and predictive tool in patients with cancer. Currently, both variant allele frequency (VAF) and number of mutant molecules per mL plasma are used as units of measurement to report those TSVs. However, it is unknown to what extent both units of measurement agree and what are the factors underlying an existing disagreement. To study the agreement between VAF and mutant molecules in current clinical studies, we analyzed 1116 TSVs from 338 patients identified with next-generation sequencing (NGS) or digital droplet PCR (ddPCR). On different study cohorts, a Deming regression analysis was performed and its 95% prediction interval was used as surrogate for the limits of agreement between VAF and number of mutant molecules per mL and to identify outliers. VAF and number of mutant molecules per mL plasma yielded greater agreement when using ddPCR than NGS. In case of discordance between VAF and number of mutant molecules per mL, insufficient molecular coverage in NGS and high cell-free DNA concentration were the main responsible factors. We propose several optimization steps needed to bring monitoring of TSVs in cell-free DNA to its full potential.

A recent study observed that numerical chromosome abnormalities in Hodgkin's disease (HD) are detected not only in morphologically abnormal Hodgkin/Reed-Sternberg cells, but also in a fraction of morphologically normal cells. However, the phenotypic constitution of these genetically abnormal, morphologically normal cells and their relationship to the malignant Hodgkin/Reed-Sternberg cells could not be established in the earlier cases studied, because of the low frequency of these cells. The present study investigated two cases of classical Hodgkin's disease containing a relatively large population of such apparently normal cells with aberrant chromosome copy numbers. The phenotype and their position within the developmental route of the malignant compartment were examined by a combined in situ hybridization and immunocytochemistry approach. Numerical abnormalities for chromosome 1 in one case and for chromosomes X, Y, and 1 in the other case were observed not only in CD30-positive Hodgkin/Reed-Sternberg cells, but also in CD30-negative, morphologically normal cells. It was shown that these genetically aberrant cells expressed the B-cell antigen CD19, thus confirming their B-cell nature. These studies indicate a relationship between the genome aberrations in these genetically abnormal, morphologically normal B-cells and the Hodgkin/Reed-Sternberg cells, suggesting that they are progenitor cells of the malignant cell fraction.

Hodgkin and Reed-Sternberg cells are considered to represent the malignant fraction in Hodgkin's disease. Several studies have shown that the Hodgkin and Reed-Sternberg cells are chromosomally abnormal, but genetic data about the morphologically normal cell population in Hodgkin's disease are very limited. This latter cell population has therefore been examined for chromosomal aberrations, using the in situ hybridization (ISH) procedure, making use of DNA probes for chromosomes 1, 7, 8, 9, 11, 12, 15, 17, and 18. Nuclei were isolated from freshly frozen (10 cases) and paraffin-embedded (16 cases) biopsy samples and 1000 nuclei per case were evaluated. The cases of Hodgkin's disease were compared with reactive lymph nodes, which show aberrant chromosome copy numbers in less than 1 per cent of the cells. Using strict scoring criteria, nuclei in the tumour were found to show an abnormal genotype, in the range of 1-12 per cent, with trisomies occurring most frequently. No characteristic numerical chromosome abnormality was observed. ISH on 4 microns thick paraffin sections of six cases of Hodgkin's disease revealed numerical aberrations for chromosome 1 in cells which appeared to be morphologically normal. The genomically abnormal nuclei did not differ in morphology or size from the nuclei of morphologically normal cells, but differed considerably in size when compared with the nuclei of Hodgkin/Reed-Sternberg cells after the ISH procedure. Three of these six cases revealed a population of apparently normal cells with an aberrant copy number which differed notably from the fraction observed in reactive lymph nodes. It is concluded, therefore, that a subset of morphologically normal cells, next to the Hodgkin/Reed-Sternberg cells, are chromosomally aberrant and may participate in the malignant cell fraction of Hodgkin's disease.

The aim of this study is to discover a gene set that can predict resistance to platinum-based chemotherapy in ovarian cancer. The study was performed on 96 primary ovarian adenocarcinoma specimens from 2 hospitals all treated with platinum-based chemotherapy. In our search for genes, 24 specimens of the discovery set (5 nonresponders and 19 responders) were profiled in duplicate with 18K cDNA microarrays. Confirmation was done using quantitative RT-PCR on 72 independent specimens (9 nonresponders and 63 responders). Sixty-nine genes were differentially expressed between the nonresponders (n=5) and the responders (n=19) in the discovery phase. An algorithm was constructed to identify predictive genes in this discovery set. This resulted in 9 genes (FN1, TOP2A, LBR, ASS, COL3A1, STK6, SGPP1, ITGAE, PCNA), which were confirmed with qRT-PCR. This gene set predicted platinum resistance in an independent validation set of 72 tumours with a sensitivity of 89% (95% CI: 0.68-1.09) and a specificity of 59% (95% CI: 0.47-0.71)(OR=0.09, p=0.026). Multivariable analysis including patient and tumour characteristics demonstrated that this set of 9 genes is independent for the prediction of resistance (p0.01). The findings of this study are the discovery of a gene signature that classifies the tumours, according to their response, and a 9-gene set that determines resistance in an independent validation set that outperforms patient and tumour characteristics. A larger independent multicentre study should further confirm whether this 9-gene set can identify the patients who will not respond to platinum-based chemotherapy and could benefit from other therapies.

We describe a fast light microscopic procedure for the simultaneous enzyme cytochemical detection of three different DNA target sequences in contrasting colors in both interphase and metaphase cell preparations. Chromosome-specific DNA probes labeled with either biotin, digoxygenin, or fluorescein were hybridized as a mixture and detected clearly and accurately by precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color), or horseradish peroxidase-tetramethylbenzidine (PO-TMB, green color) reaction, respectively. The PO-TMB reaction product was stabilized effectively by the addition of sodium tungstate to the reaction mixture, thus making the PO-TMB reaction now generally applicable to in situ hybridization (ISH). To avoid mixing of the precipitates of the two PO reactions used in the triple-color ISH method, the first detected PO activity was always completely inactivated by a mild acid treatment before the second one was applied. Finally, the cell preparations were embedded in a thin protein layer cross-linked by formaldehyde to ensure permanent stabilization of the enzyme reaction products and optimal visualization of color contrast. The triple-color ISH detection procedure could be combined with beta-galactosidase-5-bromo-4-chloro-3-indolyl-beta- D-galactoside (beta-Gal-BCIG) immunocytochemistry (ICC), leading to the simultaneous localization of multiple DNA targets and a protein target in the same cell. The described procedure may therefore be a valuable tool in the areas of cytogenetics, cell biology, and molecular pathology.