Detection of TP53 mutations in plasma of ovarian cancer and breast cancer patients

Background. The analysis of circulating tumor DNA provides wide opportunities for monitoring the results of cancer treatment. Somatic mutations in TP53 gene are present in almost all breast carcinomas developing in hereditary BRCA1 mutation carriers, as well as in the majority of high-grade serous ovarian tumors, which makes it possible to use them for effective monitoring of these diseases. The aim of the study was to analyze the content of tumor-specific TP53 mutations in plasma of patients with high-grade serous ovarian cancer (OC) and BRCA1-associated breast cancer (BC). Materials and methods. At least one plasma sample was obtained from 10 patients with OC and 7 patients with BRCA1-associated BC. The primary intratumoral status of TP53 gene was determined in the archival tumor material by targeted next generation sequencing. Digital droplet PCR was applied for testing of plasma samples for the presence of tumor-specific TP53 mutations, and in one case, BRAF V600E mutation. Results. All 8 plasma samples obtained from OC patients at the time of disease progression, before or during neoadjuvant chemotherapy, were positive for TP53 mutations. In contrast, 8 OC plasma samples obtained during remission, after surgery, or after neoadjuvant chemotherapy did not contain tumor-specific mutations. In breast cancer, circulating tumor DNA was detected in 2 of 4 samples obtained before treatment, and was not detected after the end of therapy or in remission. Conclusion. There is a good correlation between the presence of tumor-specific TP53 mutations in circulating DNA and the disease status in OC patients, therefore TP53 is a promising marker for clinical monitoring of ovarian cancer. In breast cancer, circulating tumor DNA is less abundant, therefore TP53 mutations cannot be reliably detected by digital droplet PCR in the plasma of patients with moderate disease burden.

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Patient-Specific Circulating Tumor DNA Detection during Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer

Clinical Chemistry ◽

10.1373/clinchem.2016.262337 ◽

2017 ◽

Vol 63 (3) ◽

pp. 691-699 ◽

Cited By ~ 72

Author(s):

Francesca Riva ◽

Francois-Clement Bidard ◽

Alexandre Houy ◽

Adrien Saliou ◽

Jordan Madic ◽

...

Keyword(s):

Breast Cancer ◽

Neoadjuvant Chemotherapy ◽

Minimal Residual Disease ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Residual Disease ◽

Circulating Tumor Dna ◽

Tp53 Mutations ◽

Tumor Dna ◽

Minimal Residual

Abstract BACKGROUND In nonmetastatic triple-negative breast cancer (TNBC) patients, we investigated whether circulating tumor DNA (ctDNA) detection can reflect the tumor response to neoadjuvant chemotherapy (NCT) and detect minimal residual disease after surgery. METHODS Ten milliliters of plasma were collected at 4 time points: before NCT; after 1 cycle; before surgery; after surgery. Customized droplet digital PCR (ddPCR) assays were used to track tumor protein p53 (TP53) mutations previously characterized in tumor tissue by massively parallel sequencing (MPS). RESULTS Forty-six patients with nonmetastatic TNBC were enrolled. TP53 mutations were identified in 40 of them. Customized ddPCR probes were validated for 38 patients, with excellent correlation with MPS (r = 0.99), specificity (≥2 droplets/assay), and sensitivity (at least 0.1%). At baseline, ctDNA was detected in 27/36 patients (75%). Its detection was associated with mitotic index (P = 0.003), tumor grade (P = 0.003), and stage (P = 0.03). During treatment, we observed a drop of ctDNA levels in all patients but 1. No patient had detectable ctDNA after surgery. The patient with rising ctDNA levels experienced tumor progression during NCT. Pathological complete response (16/38 patients) was not correlated with ctDNA detection at any time point. ctDNA positivity after 1 cycle of NCT was correlated with shorter disease-free (P < 0.001) and overall (P = 0.006) survival. CONCLUSIONS Customized ctDNA detection by ddPCR achieved a 75% detection rate at baseline. During NCT, ctDNA levels decreased quickly and minimal residual disease was not detected after surgery. However, a slow decrease of ctDNA level during NCT was strongly associated with shorter survival.

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Abstract 3154: Cell-free circulating tumor DNA methylation in high-grade serous ovarian cancer

10.1158/1538-7445.am2016-3154 ◽

2016 ◽

Cited By ~ 3

Author(s):

Kayleigh R. Davis ◽

Kirsty J. Flower ◽

Jane V. Borley ◽

Charlotte SM Wilhelm-Benartzi ◽

Robert Brown

Keyword(s):

Ovarian Cancer ◽

Dna Methylation ◽

Circulating Tumor Dna ◽

High Grade ◽

Serous Ovarian Cancer ◽

Tumor Dna

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Serial circulating tumor DNA samples from patients with metastatic breast cancer and BRCA1/2 mutations.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.1025 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 1025-1025

Author(s):

Katharine Collier ◽

David Tallman ◽

Zachary Weber ◽

Marcy Haynam ◽

Elizabeth J. Adams ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Clonal Evolution ◽

Metastatic Breast ◽

Circulating Tumor Dna ◽

Plasma Samples ◽

Significant Difference ◽

Platinum Chemotherapy ◽

Tumor Dna ◽

Over Time

1025 Background: Analysis of circulating tumor DNA (ctDNA) over time allows non-invasive evaluation of tumor genomic evolution. We characterize changes in tumor fraction (TFx), somatic copy number alterations (SCNAs), and somatic mutations (muts) over time in patients (pts) with BRCA1/2 muts and metastatic breast cancer (mBC) who received a PARP inhibitor (PARPi) or platinum chemotherapy. Specifically, we seek to identify the frequency of BRCA1/2 reversion muts. Methods: Pts with mBC and germline or somatic BRCA1/2 muts were identified on a banking protocol of prospectively-collected serial samples of blood and plasma. Control pts without a BRCA1/2 mut were matched 2:1 by age and hormone receptor (HR) status. Ultra-low-pass whole genome sequencing (ULPWGS) with 0.1x depth was performed on all plasma samples (n = 103) and the ichorCNA algorithm was used to determine TFx and SCNAs. Targeted panel sequencing (TPS) of 402 cancer-related genes was performed at 10,000x depth on plasma samples, and one blood sample per pt. The panel includes BRCA1/2 and 38 other DNA damage repair (DDR) genes. Somatic muts were identified by joint calling with Mutect2 across plasma timepoints with paired pt normal blood. Germline variant calling from TPS on blood with HaplotypeCaller was used to confirm germline muts in BRCA1/2.Results: We identified 10 pts with mBC with a germline (n = 7) or somatic (n = 3) BRCA1 (n = 2) or BRCA2 (n = 8) mut and banked blood and plasma samples at 3-9 timepoints at a median of 8 weeks apart (range 1-43). The control cohort of 20 pts with mBC and wildtype BRCA1/2 was well matched by age and HR status. All pts with BRCA1/2 muts received a PARPi and/or platinum chemotherapy at some point during sample collection. Half of control pts received platinum chemotherapy. Germline BRCA1/2 muts were confirmed in all 7 pts with known germline muts. Among the BRCA1/2 mut cohort, median TFx was 0.04 (range 0-0.57) with 20% of samples having TFx > 0.10. A median of 1.5 (range 0-39) somatic muts per pt were found in DDR genes. Four pts (40%) had secondary non-reversion muts in BRCA1/2. A reversion mut of a germline BRCA2 mut, restoring the open reading frame of BRCA2, was discovered at the last timepoint from 1 pt while receiving carboplatin. A germline BRCA1/2 reversion mut in this cohort occurred in 2.3% of samples, 14.3% of pts. There was no significant difference in the percent of genome with a SCNA between the first and last time point, nor before and after PARPi/platinum. The somatic mut landscape and clonal evolution of TPS using PyClone will be presented. Conclusions: Evaluation of serial ctDNA samples for TFx, SCNAs, and somatic muts from banked plasma and blood from pts with mBC is feasible. The frequency of reversion muts in BRCA1/2 was low, suggesting that either their incidence is low or ctDNA TPS is not sensitive enough to detect them. Secondary non-reversion muts in BRCA1/2 and other somatic DDR muts were more common. SCNAs were stable over time.

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Abstract 4587:ESR1methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer

10.1158/1538-7445.am2018-4587 ◽

2018 ◽

Author(s):

Lydia Giannopoulou ◽

Sofia Mastoraki ◽

Areti Strati ◽

Issam Chebouti ◽

Kitty Pavlakis ◽

...

Keyword(s):

Ovarian Cancer ◽

Circulating Tumor Dna ◽

High Grade ◽

Serous Ovarian Cancer ◽

Primary Tumors ◽

Tumor Dna

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Prognostic and predictive value of circulating tumor DNA during neoadjuvant chemotherapy for triple negative breast cancer

Scientific Reports ◽

10.1038/s41598-020-71236-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Luca Cavallone ◽

Adriana Aguilar-Mahecha ◽

Josiane Lafleur ◽

Susie Brousse ◽

Mohammed Aldamry ◽

...

Keyword(s):

Breast Cancer ◽

Neoadjuvant Chemotherapy ◽

Triple Negative Breast Cancer ◽

Predictive Value ◽

Triple Negative ◽

Residual Tumor ◽

Complete Response ◽

Circulating Tumor Dna ◽

Droplet Pcr ◽

Tumor Dna

Abstract Response to neoadjuvant chemotherapy (NAC) in triple negative breast cancer (TNBC) is highly prognostic and determines whether adjuvant chemotherapy is needed if residual tumor is found at surgery. To evaluate the predictive and prognostic values of circulating tumor DNA (ctDNA) in this setting, we analyzed tumor and serial bloods from 26 TNBC patients collected prior, during, and after NAC. Individual digital droplet PCR assays were developed for 121 variants (average 5/patient) identified from tumor sequencing, enabling ctDNA detection in 96% of patients at baseline. Mutant allele frequency at baseline was associated with clinical characteristics. Levels drastically fell after one cycle of NAC, especially in patients whose tumors would go on to have a pathological complete response (pCR), but then rose significantly before surgery in patients with significant residual tumor at surgery (p = 0.0001). The detection of ctDNA early during treatment and also late at the end of NAC before surgery was strongly predictive of residual tumor at surgery, but its absence was less predictive of pCR, especially when only TP53 variants are considered. ctDNA detection at the end of neoadjuvant chemotherapy indicated significantly worse relapse-free survival (HR = 0.29 (95% CI 0.08–0.98), p = 0.046), and overall survival (HR = 0.27 95% CI 0.075–0.96), p = 0.043). Hence, individualized multi-variant ctDNA testing during and after NAC prior to surgery has prognostic and predictive value in early TNBC patients.

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Cell-free tumor DNA in peri-operative blood samples from advanced high-grade serous ovarian cancer patients with and without complete resection.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.5555 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 5555-5555 ◽

Cited By ~ 1

Author(s):

Florian Heitz ◽

Sotirios Lakis ◽

Philipp Harter ◽

Jalid Sehouli ◽

Jatin Talwar ◽

...

Keyword(s):

Ovarian Cancer ◽

Residual Disease ◽

Complete Resection ◽

High Grade ◽

Serous Ovarian Cancer ◽

Debulking Surgery ◽

Tp53 Mutations ◽

Post Surgery ◽

Tumor Dna

5555 Background: Surgical outcome is an important prognostic factor in advanced high-grade serous ovarian cancer (HGOC). Intra-operative determination of residual disease may be prone to subjective bias. Methods: We prospectively collected and analyzed tumor tissue and plasma from 21 pts with FIGO IIIC-IV HGOC. All tumor specimen showed loss of TP53 in immunohistochemistry. Tumor DNA from tissue and peri-operative cell-free DNA at baseline (BL) and at d(ays) 1, d4 and d10 were subjected to comprehensive hybrid-capture based next-generation sequencing (NEO New Oncology GmbH, Cologne, Germany). Results: The initial cohort comprised 10 pts without (TR>0) and 11 pts with complete resection TR0, with a total of 43 somatic genomic alterations. TP53 was mutated in 20/21 (95.2%) of tissue samples and in 15/21 (71.4%) corresponding plasma samples at BL. Therefore, TP53 mutations were used as a molecular marker of circulating tumor DNA levels post-surgery. In the remaining 5 BL cases the TP53 mutations were either not present in plasma (N= 3) or rested below the detection limit (N= 2). In d1, d4 and d10 post-surgical samples, the sample specific TP53 mutations were detected in 12 out of 15, 10 out of 14 and in 7 out of 13 cases, respectively. TP53variant allele frequency (VAF) did not differ among TR0 and TR>0 at BL (mean= 1.91 versus 1.73, Mann-Whitney test p = 0.86) whereas it was significantly lower for TR0 at d1 (mean= 0.06 versus 2,06; p value= 0.002), d4 (mean= 0.07 versus 1.8; p= 0.04) and d10 (mean VAF= 0 versus 1.04; p= 0.008). Five out of 9 TR>0 cases showed at least one increase in VAF between d0 and any additional time-point. In 8 out of 9 mutations remained detectable at d10 (VAF: 0.076 – 3.26). By contrast, TR0 cases showed consistent reduction in VAF throughout the follow-up period which maximized at d10. Conclusions: Liquid biopsycan efficiently detect somatic mutations in the cfDNA of pts with HGOC following debulking surgery. Variation of the TP53 VAF in sequential post-surgical samples appears to be restricted to TR>0 cases, whereas in TR0 pts VAF progressively decreases. Liquid biopsy may hold promise as a tool for the objective determination of residual disease after debulking surgery.

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Analysis of circulating tumor DNA (ctDNA) in pseudoprogression in anti-PD1 treated metastatic melanoma (MM).

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.9546 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 9546-9546

Author(s):

Jenny HJ Lee ◽

Georgina V. Long ◽

Alexander M. Menzies ◽

Alexander David Guminski ◽

Richard Kefford ◽

...

Keyword(s):

Disease Progression ◽

Circulating Tumor Dna ◽

The Other ◽

Clinical Progression ◽

Radiological Progression ◽

Digital Droplet Pcr ◽

Droplet Pcr ◽

True Progression ◽

Tumor Dna

9546 Background: We have previously shown that undetectable ctDNA either at baseline or during therapy predicted response in mm patients (pts) treated with anti-PD1 antibodies (aPD1). Pseudoprogression, defined as radiological progression prior to response, occurs in 8% of pts treated with aPD1. We sought to determine if ctDNA could differentiate pseudoprogression from true progression, defined as continued clinical or radiological disease progression. Methods: Between July 2014 and May 2016, pts receiving aPD1 had serial bloods for ctDNA. Included pts either had RECIST PD at first restaging or early clinical progression. Those with untreated brain metastases were excluded from the analysis. ctDNA was quantified using digital droplet PCR for mutations (BRAF/NRAS) at baseline and during the first 12 wks of treatment. Based on our prior studies, ctDNA results were grouped in to ‘favorable’ and ‘unfavorable’ ctDNA profiles (see Table), and these were compared in pts with true and pseudoprogression. Results: 29 pts were included, 28 with RECIST PD at first restaging and one with early clinical progression. 9 (31%) pts had a subsequent RECIST PR or SD and were considered pseudoprogression and 20 (69%) had true progression. Of the pseudoprogressors, 7/9 pts remained in response with a median follow-up of 20 months (mths). 2/9 pts had disease progression at 7 and 18 mths, with ctDNA that remained detectable with a > 10-fold decrease during treatment in both patients. Of those with true progression and a favourable profile, 1 had a > 10-fold decrease in ctDNA by wk 12 and was switched to MAPK therapy prior to further imaging, and the other had an undetectable ctDNA at wk 6 which increased again at wk 12. The latter pt had a new lesion on first restaging CT scan despite PR in all existing lesions with true PD on second restaging at wk 24. Conclusions: ctDNA in patients with mm at baseline and early on aPD1 treatment differentiates pseudo from true progression. [Table: see text]

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