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TP53 Mutations in Serum Circulating Cell-Free Tumor DNA As Longitudinal Biomarker for High-Grade Serous Ovarian Cancer.

ABSTRACT: The aim of this study was to determine an optimal workflow to detect TP53 mutations in baseline and longitudinal serum cell free DNA (cfDNA) from high-grade serous ovarian carcinomas (HGSOC) patients and to define whether TP53 mutations are suitable as biomarker for disease. TP53 was investigated in tissue and archived serum from 20 HGSOC patients by a next-generation sequencing (NGS) workflow alone or combined with digital PCR (dPCR). AmpliSeq™-focused NGS panels and customized dPCR assays were used for tissue DNA and longitudinal cfDNAs, and Oncomine NGS panel with molecular barcoding was used for baseline cfDNAs. TP53 missense mutations were observed in 17 tissue specimens and in baseline cfDNA for 4/8 patients by AmpliSeq, 6/9 patients by Oncomine, and 4/6 patients by dPCR. Mutations in cfDNA were detected in 4/6 patients with residual disease and 3/4 patients with disease progression within six months, compared to 5/11 patients with no residual disease and 6/13 patients with progression after six months. Finally, mutations were detected at progression in 5/6 patients, but not during chemotherapy. NGS with molecular barcoding and dPCR were most optimal workflows to detect TP53 mutations in baseline and longitudinal serum cfDNA, respectively. TP53 mutations were undetectable in cfDNA during treatment but re-appeared at disease progression, illustrating its promise as a biomarker for disease monitoring.

SUBMITTER: Vitale SR

PROVIDER: S-EPMC7175353 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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TP53 Mutations in Serum Circulating Cell-Free Tumor DNA As Longitudinal Biomarker for High-Grade Serous Ovarian Cancer.

Vitale Silvia R SR Groenendijk Floris H FH van Marion Ronald R Beaufort Corine M CM Helmijr Jean C JC Dubbink Hendrikus Jan HJ Dinjens Winand N M WNM Ewing-Graham Patricia C PC Smolders Ramon R van Doorn Helena C HC Boere Ingrid A IA Berns Els M J J EMJJ Helleman Jozien J Jansen Maurice P H M MPHM

Biomolecules 20200307 3

The aim of this study was to determine an optimal workflow to detect TP53 mutations in baseline and longitudinal serum cell free DNA (cfDNA) from high-grade serous ovarian carcinomas (HGSOC) patients and to define whether TP53 mutations are suitable as biomarker for disease. TP53 was investigated in tissue and archived serum from 20 HGSOC patients by a next-generation sequencing (NGS) workflow alone or combined with digital PCR (dPCR). AmpliSeq™-focused NGS panels and custom ...[more]

PMID: 32156073

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Project description:BackgroundHigh grade serous ovarian cancer is characterised by high initial response to chemotherapy but poor outcome in the long term due to acquired resistance. One of the main genetic features of this disease is TP53 mutation. The majority of TP53 mutated tumors harbor missense mutations in this gene, correlated with p53 accumulation. TP53 null tumors constitute a specific subgroup characterised by nonsense, frameshift or splice-site mutations associated to complete absence of p53 expression. Different studies show that this kind of tumors may have a worse prognosis than other TP53 mutated HGSC.MethodsIn this study, we sought to characterise the intra-tumor heterogeneity of a TP53 null HGSC consisting of six primary tumor samples, two intra-pelvic and four extra-pelvic recurrences using exome sequencing and comparative genome hybridisation.ResultsSignificant heterogeneity was found among the different tumor samples, both at the mutational and copy number levels. Exome sequencing identified 102 variants, of which only 42 were common to all three samples; whereas 7 of the 18 copy number changes found by CGH analysis were presented in all samples. Sanger validation of 20 variants found by exome sequencing in additional regions of the primary tumor and the recurrence allowed us to establish a sequence of the tumor clonal evolution, identifying those populations that most likely gave rise to recurrences and genes potentially involved in this process, like GPNMB and TFDP1. Using functional annotation and network analysis, we identified those biological functions most significantly altered in this tumor. Remarkably, unexpected functions such as microtubule-based movement and lipid metabolism emerged as important for tumor development and progression, suggesting its potential interest as therapeutic targets.ConclusionsAltogether, our results shed light on the clonal evolution of the distinct tumor regions identifying the most aggressive subpopulations and at least some of the genes that may be implicated in its progression and recurrence, and highlights the importance of considering intra-tumor heterogeneity when carrying out genetic and genomic studies, especially when these are aimed to diagnostic procedures or to uncover possible therapeutic strategies.

Project description:BackgroundCirculating tumour DNA (ctDNA) carrying tumour-specific sequence alterations may provide a minimally invasive means to dynamically assess tumour burden and response to treatment in cancer patients. Somatic TP53 mutations are a defining feature of high-grade serous ovarian carcinoma (HGSOC). We tested whether these mutations could be used as personalised markers to monitor tumour burden and early changes as a predictor of response and time to progression (TTP).Methods and findingsWe performed a retrospective analysis of serial plasma samples collected during routine clinical visits from 40 patients with HGSOC undergoing heterogeneous standard of care treatment. Patient-specific TP53 assays were developed for 31 unique mutations identified in formalin-fixed paraffin-embedded tumour DNA from these patients. These assays were used to quantify ctDNA in 318 plasma samples using microfluidic digital PCR. The TP53 mutant allele fraction (TP53MAF) was compared to serum CA-125, the current gold-standard response marker for HGSOC in blood, as well as to disease volume on computed tomography scans by volumetric analysis. Changes after one cycle of treatment were compared with TTP. The median TP53MAF prior to treatment in 51 relapsed treatment courses was 8% (interquartile range [IQR] 1.2%-22%) compared to 0.7% (IQR 0.3%-2.0%) for seven untreated newly diagnosed stage IIIC/IV patients. TP53MAF correlated with volumetric measurements (Pearson r = 0.59, p < 0.001), and this correlation improved when patients with ascites were excluded (r = 0.82). The ratio of TP53MAF to volume of disease was higher in relapsed patients (0.04% per cm3) than in untreated patients (0.0008% per cm3, p = 0.004). In nearly all relapsed patients with disease volume > 32 cm3, ctDNA was detected at ?20 amplifiable copies per millilitre of plasma. In 49 treatment courses for relapsed disease, pre-treatment TP53MAF concentration, but not CA-125, was associated with TTP. Response to chemotherapy was seen earlier with ctDNA, with a median time to nadir of 37 d (IQR 28-54) compared with a median time to nadir of 84 d (IQR 42-116) for CA-125. In 32 relapsed treatment courses evaluable for response after one cycle of chemotherapy, a decrease in TP53MAF of >60% was an independent predictor of TTP in multivariable analysis (hazard ratio 0.22, 95% CI 0.07-0.67, p = 0.008). Conversely, a decrease in TP53MAF of ?60% was associated with poor response and identified cases with TTP < 6 mo with 71% sensitivity (95% CI 42%-92%) and 88% specificity (95% CI 64%-99%). Specificity was improved when patients with recent drainage of ascites were excluded. Ascites drainage led to a reduction of TP53MAF concentration. The limitations of this study include retrospective design, small sample size, and heterogeneity of treatment within the cohort.ConclusionsIn this retrospective study, we demonstrated that ctDNA is correlated with volume of disease at the start of treatment in women with HGSOC and that a decrease of ?60% in TP53MAF after one cycle of chemotherapy was associated with shorter TTP. These results provide evidence that ctDNA has the potential to be a highly specific early molecular response marker in HGSOC and warrants further investigation in larger cohorts receiving uniform treatment.

Project description:While TP53 mutation is widely considered to be a defining feature of tubo-ovarian high-grade serous carcinoma (HGSC), rare TP53-mutation-negative cases have been reported. To gain further insight into this rare subset, a retrospective review was conducted on 25 TP53-wildtype tubo-ovarian HGSCs, constituting 2.5% of 987 HGSCs profiled by the MSK-IMPACT sequencing platform. Consistent with serous differentiation, positive staining for Pax8 and WT1 was present in virtually all TP53-wildtype HGSCs. Other characteristic features of HGSC, such as serous tubal intraepithelial carcinoma, or genetic alterations of CCNE1 and BRCA1/2 were identified in these tumors, furthering supporting their classification as bona fide HGSC, despite lacking TP53 mutations. Overall, the level of chromosomal instability of TP53-wildtype HGSCs was intermediate between low-grade serous carcinoma (LGSC) and TP53-mutated HGSC. Morphologic assessment by observers blinded to mutation status revealed a significant subset of tumors with Grade 2 nuclear atypia (which exceeds the degree of atypia allowed for LGSC, but less than typically encountered for HGSC) combined with micropapillary features (6/19, 32%, chemotherapy-naive TP53-wildtype HGSCs compared to 0/21, 0%, TP53-mutated HGSCs; p = 0.007). Some TP53-wildtype HGSCs harbored driver mutations in KRAS (n = 3), BRAF (n = 1) or NRAS (n = 2). Overall, 10 (40%) cases had "LGSC-like" morphology (i.e., Grade 2 nuclear atypia and micropapillary features) and/or RAS/RAF mutation, and most of these showed a wildtype p53 pattern of expression by immunohistochemistry (7/9, 78%). The remaining TP53-wildtype HGSCs (n = 15, 60%) exhibited severe nuclear atypia (Grade 3) and were morphologically indistinguishable from conventional TP53-mutated HGSC. Despite lacking genetic alterations of TP53, these "usual HGSC-like" tumors often showed evidence of p53 dysfunction, including downregulation of expression ('null' or equivocal p53 staining in 9/14, 64%) or MDM2 amplification (n = 2). Our results support the existence of TP53-wildtype HGSCs, which comprise a heterogeneous group of tumors which may arise via distinct pathogenic mechanisms.

Dataset Information

TP53 Mutations in Serum Circulating Cell-Free Tumor DNA As Longitudinal Biomarker for High-Grade Serous Ovarian Cancer.

Publications

<i>TP53</i> Mutations in Serum Circulating Cell-Free Tumor DNA As Longitudinal Biomarker for High-Grade Serous Ovarian Cancer.

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