The emergence of cisplatin (CDDP) resistance is the main cause of treatment failure and death in patients with testicular germ cell tumors (TGCT), but its biologic background is poorly understood. To study the molecular basis of CDDP resistance in TGCT we prepared and sequenced CDDP-exposed TGCT cell lines as well as 31 primary patients’ samples. Long-term exposure to CDDP increased the CDDP resistance 10 times in the NCCIT cell line, while no major resistance was achieved in Tera-2. Development of CDDP resistance was accompanied by changes in the cell cycle (increase in G1 and decrease in S-fraction), increased number of acquired mutations, of which 3 were present within ATRX gene, as well as changes in gene expression pattern. Copy number variation analysis showed, apart from obligatory gain of 12p, several other large-scale gains (chr 1, 17, 20, 21) and losses (chr X), with additional more CNVs found in CDDP-resistant cells (e.g., further losses on chr 1, 4, 18, and gain on chr 8). In the patients’ samples, those who developed CDDP resistance and died of TGCT (2/31) showed high numbers of acquired aberrations, both SNPs and CNVs, and harbored mutations in genes potentially relevant to TGCT development (e.g., TRERF1, TFAP2C in one patient, MAP2K1 and NSD1 in another one). Among all primary tumor samples, the most commonly mutated gene was NSD1, affected in 9/31 patients. This gene encoding histone methyl transferase was also downregulated and identified among the 50 most differentially expressed genes in CDDP-resistant NCCIT cell line. Interestingly, 2/31 TGCT patients harbored mutations in the ATRX gene encoding a chromatin modifier that has been shown to have a critical function in sexual differentiation. Our research newly highlights its probable involvement also in testicular tumors. Both findings support the emerging role of altered epigenetic gene regulation in TGCT and CDDP resistance development.
4. Minard-Colin V, Auperin A, Pillon M, et al. Results of the randomized Intergroup trial Inter-B-NHL Ritux 2010 for children and adolescents with high-risk B-cell non-Hodgkin lymphoma (B-NHL) and mature acute leukemia (B-AL): Evaluation of rituximab (R) efficacy in addition to standard LMB chemotherapy (CT) regimen [abstract]. J Clin Oncol. 2016;34(15). Abstract 10507. 5. Cairo M, Auperin A, Perkins SL, et al. Overall survival of children and adolescents with mature B cell non-Hodgkin lymphoma who had refractory or relapsed disease during or after treatment with FAB/LMB 96: a report from the FAB/LMB 96 study group.
e17519 Background: The clinical management of testicular germ cell tumors (TGCT) has not changed much for decades; most importantly, novel prognostic factors indicating the need of adjuvant chemotherapy and factors related to the development of cisplatin resistance would be needed. Circulating free tumor DNA (cfDNA) is an easily available and valuable source of genetic material, which may bring clinically relevant information. Methods: After ethical committee approval and patient informed consent, peripheral blood (PB) samples were taken from TGCT patients at the diagnosis (after orchidectomy), after 2 cycles of chemotherapy, at the end of treatment, and at relapse or disease progression, if applicable. Clinical data of all patients were recorded. The PB samples were processed immediately after collection, plasma was separated by centrifugation, cfDNA was extracted by QIAmp Circulating Nucleic Acid kits (Qiagen), its quality and quantity was assessed by capillary electrophoresis (Agilent) and qPCR for a house-keeping gene. Selected samples were subjected to whole exome sequencing using SureSelectXT HS + Human All Exon v6 kits (Agilent) on NextSeq (Illumina) platform, together with the corresponding primary testicular tumor and peripheral blood mononuclears as a germ-line control. Statistical analyses were performed using non-parametric tests. Results: Sixty-three samples of 41 patients have been analyzed. The median amount of detected cfDNA did not significantly differ from non-malignant controls, was similar in seminoma and non-seminoma pts, but was significantly higher (p = 0.01) in pts with disease progression. In pts with elevated cfDNA levels, these decreased after 2 and 4 cycles of chemotherapy. In 5 sequenced pts, molecular aberrations (somatic missense or frameshift mutations) were found in genes CDC27 (2 pts), RBMX (4 pts), TPTE2 (3 pts), and TSPAN16 (1 pt). These aberrations were also detected in primary tumors but with lower frequencies, and were not present in germ-line DNA. CDC27 mutations have been described in TGCT previously. The other genes have not yet been linked to TGCT, although their role in spermatogenesis and cell proliferation is well known. The presence of mitochondrial DNA was not detected in cfDNA. Conclusions: High levels of cfDNA are detectable in pts with disease progression where they reflect the total tumor load; the molecular analysis of cfDNA revealed novel aberrations that may play a role in TGCT development. Supported by grants MH CZ - DRO00064190TN, CDRO00064203FNM and MEYS NPU I LO1604
Objective: The evaluation of quantitative fluorescence PCR (QF-PCR) and single nucleotide polymorphism array (SNP array) analysis for the identification of chromosomal abnormalities in products of conception (POC). Materials and methods: A total of 1,094 POC samples were processed at Gennet in the years 2018–2020. Chromosomal aneuploidies were tested by QF-PCR using a Omnibor set (STR markers 13, 18, 21, X a Y), SAB-I set (STR markers 2, 7, 15, 16, 22), SAB-II set (from November 2019, STR markers 4, 6, 14) followed by SNP array analysis (Illumina) on samples with a negative QF-PCR result. All POC samples were tested for maternal contamination. Results: After exclusion of maternal contamination (32% samples) the total number of 742 POC samples were tested by QF-PCR. Chromosomal aneuploidies were found in 273 POC samples (36.8%). Then, 469 QF-PCR negative POC samples were tested by SNP array analysis. Normal female/ male profi le was confirmed in 402 samples (85.7%) and chromosomal aneuploidies and chromosomal aberrations (deletion/ duplication > 10 Mb) in 51 samples (10.9%). Microdeletion/ microduplication was found in 16 POC samples (3.4%), two were classified as pathogenic variants and 14 as variants of unknown significance. In a group of women > 35 years of age, statistically significant increase of the chromosomal abnormalities was confirmed. No statistically significant difference between the in vitro fertilization group and the group of spontaneous conception was found. Conclusion: The application of the molecular work-up based on the stepwise use of QF-PCR and SNP array clarifies the cause of the abortion in 43% POC samples. The overall detection rate in the I. trimester was 50.4%. Key words: aborted fetus – QF-PCR – SNP-array – chromosomal aberration – aneuploidy
Introduction MRD is an important predictor of outcome in childhood ALL. Since 2000, MRD detected by quantitative PCR (qPCR) for immunoglobulin and T-cell receptor gene rearrangements with a minimal sensitivity of 1E-04 has been used for risk group stratification in pediatric BFM trials. Next generation sequencing (NGS) permits rapid parallel sequencing of large numbers of DNA segments. It can overcome most of the limitations of qPCR: it allows highly specific molecular detection of MRD without laborious optimization of patient-specific assays. Moreover it enables not only monitoring of malignant clone but also shows the picture of entire immune background. Aims To develop an assay for immunoglobulin heavy chain (IgH) rearrangements detection on Ion Torrent PGM/Ion Proton platforms and compare the MRD levels with qPCR at BFM stratification timepoints. Methods Two round PCR was used for library preparation. Libraries were created from 450ng (equivalent of 70,000 DNA copies) of bone marrow DNA and 50ng of Human Genomic DNA (Roche). In the first round of PCR rearranged IgH genes were amplified using IGH FR3 BIOMED-2 primers (van Dongen, Leukemia 2003). In the second round the sequencing adapters and multiplex identifiers were added. Sequencing was performed on Ion Torrent PGM/Ion Proton sequencers using a 200bp chemistry. We developed a bioinformatics algorithm for detection of reads with known clonal V-D-J rearrangements within the resulting fastq files. For validation of the assay we sequenced 1E-1 to 1E-5 dilutions of diagnostic samples from 2 patients in multiplicates. The results show that the assay gives reproducible quantitative results up to 1E-4 dilution. Results We sequenced 183 samples from 67 patients (52×day 15, 65×day 33, 66×day 78) with childhood ALL treated according to AIEOP-BFM ALL 2000 protocol with the median coverage 587,406 reads per sample. Eighty-three (45.4%) samples were negative by both methods. Fifteen (8.2%) samples were positive by NGS and negative by qPCR and 14 (7.7%) samples were positive by qPCR and negative by NGS. All the discordant samples had MRD levels below the sensitivity of both methods. The overall correlation of all double positive and double negative samples was very good (R2=0.93). If risk group stratification based on NGS results would be performed, 8 patients would be classified as intermediate risk (IR) instead of standard risk (SR) (one of whom relapsed) and 8 patients as SR instead of IR. One patient would be relocated from IR to slow early responders (SER) group, and two patients from SER to IR (one of them relapsed). One patient who relapsed would be classified as high risk (HR) instead of SER. All 5 patients who were MRD negative at d15 by NGS remained MRD negative in later timepoints and none of them relapsed. Discussion We present a cost-effective and widely adoptable NGS-based method that provides clinically relevant results in childhood ALL. NGS has a great potential to reduce the laboriousness associated with patient-specific qPCR analysis and to speed up the process of MRD detection. The sensitivity of both methods is comparable when ~500ng of DNA is used. The majority of the differences were in the samples with MRD levels below 1E-4 and most of treatment stratification changes occurred between SR/IR. However, the different stratification mostly concerned patients who did not relapse. The sensitivity of NGS could be improved if more DNA was analyzed. However, the benefit of increased MRD sensitivity is questionable due to possible overtreatment of patients with very low MRD loads after induction treatment. “Online” identification of d15 MRD negative patients reported as having an excellent prognosis previously is possible only by NGS, because optimization of patient-specific qPCR takes several weeks. The addition of 10% polyclonal DNA seems to solve the problem of MRD overestimation by NGS in samples with B-cell aplasia. At present, the main drawback of the Ig/TCR-exploring NGS methods is lack of standardization both in the experimental setting and in data analysis. Therefore, recently a European network, the EuroClonality NGS Consortium, has been formed to standardize the whole workflow of analytics, pre-analytics and bioinformatics not only for MRD quantification but also for clonality assessment in lymphoid neoplasms and for repertoire analysis. Supported by IGA NT14343, IGA NT12397, IGA NT13462-4 and GAUK 394214. Disclosures No relevant conflicts of interest to declare.
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