DNA Of Breast Cancer Patients Research Articles

Clinical Significance of Circulating Cell-Free Mitochondrial DNA in Breast Cancer Patients

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The Clinical Utility of Droplet Digital PCR for Profiling Circulating Tumor DNA in Breast Cancer Patients.

Breast cancer is the most common cancer affecting women worldwide. It is a malignant and heterogeneous disease with distinct molecular subtypes, which has prognostic and predictive implications. Circulating tumor DNA (ctDNA), cell-free fragmented tumor-derived DNA in blood plasma, is an invaluable source of specific cancer-associated mutations and holds great promise for the development of minimally invasive diagnostic tests. Furthermore, serial monitoring of ctDNA over the course of systemic and targeted therapies not only allows unparalleled efficacy assessments but also enables the identification of patients who are at risk of progression or recurrence. Droplet digital PCR (ddPCR) is a powerful technique for the detection and monitoring of ctDNA. Due to its relatively high accuracy, sensitivity, reproducibility, and capacity for absolute quantification, it is increasingly used as a tool for managing cancer patients through liquid biopsies. In this review paper, we gauge the clinical utility of ddPCR as a technique for mutational profiling in breast cancer patients and focus on HER2, PIK3CA, ESR1, and TP53, which represent the most frequently mutated genes in breast cancers.

Analysis of plasma HER2 copy number in cell-free DNA of breast cancer patients: a comparison with HER2 extracellular domain protein level in serum

HER2 (human epidermal growth factor receptor 2) status has been evaluated in breast cancer (BC) tissues by immunohistochemistry or in situ hybridization. We evaluated HER2 copy number (CN) assay in plasma cell-free DNA (cfDNA) from blood samples and compared it with protein measurements of HER2 extracellular domain (ECD) in serum. Serum HER2-ECD levels were measured by chemi-luminescence immunoassay using anti-HER2 monoclonal antibodies. Analyses were performed on 120 cases of primary BC, 30 cases of metastatic BC and 34 cases treated by neoadjuvant chemotherapy (NAC). This study was approved by Medical Research Review Advancement No. 1857 for Kumamoto University. There was a positive correlation between HER2-CN ratios and HER2-ECD levels, in primary (n = 54) and metastatic (n = 30) HER2-positive BC (P = 0.003 and P < 0.001, respectively). HER2-ECD levels were significantly higher in patients with a larger number of metastatic sites (P = 0.02). The usefulness of HER2 levels in discriminating primary and metastatic HER2-positive BC evaluated by ROC curve analysis was better in the HER2-ECD assay than in the HER2-CN assay. In 34 patients who received NAC, there was a small decrease in HER2-CN ratios between before and after NAC (P = 0.10), while there was an obvious decrease in HER2-ECD levels between before and after NAC (P < 0.001). Compared to HER2-ECD levels, the clinical usefulness of HER2-CN ratio was somewhat inferior. Improved measurement methods and further examination of the association with long-term prognosis and the response to anti-HER2 treatment analyzed by HER2-CN and HER2-ECD assay are required.

Epigenetic markers-based breast cancer early detection method development

Relevance: According to the International Agency for Research on Cancer (IARС), breast cancer ranks&#x0D; 1st-2nd among other cancers globally [1], including&#x0D; Kazakhstan [2]. In Kazakhstan, the annual growth&#x0D; in breast cancer incidence exceeds 26.6%. In 2018-&#x0D; 2019, breast cancer was the 3rd most common cause&#x0D; of cancer death in Kazakhstan, accounting for 8.7-8.1&#x0D; per cent, respectively. Early detection of breast cancer&#x0D; remains an acute issue. In particular, early detection&#x0D; should be improved. Epigenetic studies of cancer patients confirm that epigenetic biomarkers could be&#x0D; used as early cancer diagnostic markers, including&#x0D; breast cancer.&#x0D; The purpose was to find specific diagnostic markers by methylation profiling of peripheral blood mononuclear fraction DNA in breast cancer patients.&#x0D; Results: Plasma samples of the Kazakhstani population with breast cancer possessed mononuclear&#x0D; fraction methylation markers in CpG islets associated&#x0D; with JAM3, C17orf64, MSC, and C7orf51 genes and the&#x0D; CpG islet associated with the intragenic region of the 5,&#x0D; chr5: 77,208,034-77,329,434 chromosome, which were&#x0D; missing in healthy individuals. These biomarkers allow&#x0D; differentiating breast cancer from other cancers with a&#x0D; specificity of 0.91 and a sensitivity of 0.94 compared to&#x0D; methylation data from open DNA methylation databases (for Illumina 450K): TCGA, GSE40279, GSE61496,&#x0D; GSE76269 и GSE66836.&#x0D; Conclusion: Early breast cancer detection method using peripheral blood mononuclear fraction DNA&#x0D; methylation profile, namely in CpG islets associated&#x0D; with JAM3, C17orf64, MSC, and C7orf51 genes and the&#x0D; CpG islet associated with the intragenic region of the&#x0D; 5, chr5: 77,208,034-77,329,434 chromosome is enough&#x0D; specific and sensitive to use it in breast cancer screening

Comparison of Circulating Tumour DNA and Extracellular Vesicle DNA by Low-Pass Whole-Genome Sequencing Reveals Molecular Drivers of Disease in a Breast Cancer Patient.

There is increasing recognition of circulating tumour DNA (ctDNA) as a non-invasive alternative to tumour tissue for the molecular characterisation and monitoring of disease. Recent evidence suggests that cancer-associated changes can also be detected in the DNA contained within extracellular vesicles (EVs). As yet, there has been limited investigation into the relationship between EV DNA and ctDNA, and no studies have examined the EV DNA of breast cancer patients. The aim of this study was to use low-pass whole-genome sequencing to identify copy number variants (CNVs) in serial samples of both ctDNA and EV DNA from a patient with breast cancer. Of the 52 CNVs identified in tumour DNA, 36 (69%) were detected in at least one ctDNA sample and 13 (25%) in at least one EV DNA sample. The number of detectable variants in ctDNA and EV DNA increased over the natural history of the patient’s disease, which was associated with progression to cerebral metastases. This case study demonstrates that, while CNVs are detectable in patient EV DNA, ctDNA has greater sensitivity than EV DNA for serial monitoring of breast cancer.

Diagnostic value of cfDNA and long fragment DNA in patients with breast cancer

To investigate the diagnostic value of cell-free DNA (cfDNA) and long fragment DNA in breast cancer patients.Female patients with breast cancer (n = 80) were recruited over one year for this study, and served as an observation group. The control group consisted of 50 normal, healthy females. Plasma levels of cfDNA and long fragment DNA were determined a day before treatment, 7 days after treatment, and on the 20th day of treatment. The levels of cfDNA and long fragment DNA in breast cancer patients before treatment were significantly higher than those of the control group (p&lt;0.05). Patients cfDNA and long fragment DNA levels 7 days after treatment were not significantly different from the corresponding values at 1 day before treatment (p&gt;0.05), but they decreased significantly on the 20th day of treatment, when compared with levels before treatment (p&lt;0.05). Before treatment, the optimal cut-off point for cfDNA in patients' peripheral blood, sensitivity, specificity and accuracy were 12.25ng/mL, 79.12%, 86.15%, and 73.32%, respectively. The area under the ROC curve (AUC) was 0.865 (95% CI = 0.754-0.903). Close monitoring of cfDNA levels in peripheral blood of breast cancer patients in real-time can be used for early diagnosis of the disease.

Presence of human papillomavirus DNA in breast cancer patients: SERUM and tissue analysis.

e12582 Background: It has been demonstrated an increased risk of breast cancer (BC) incidence in patients with previous cervical dysplasia, suggesting a role of human papillomavirus (HPV) of cervical lesions in the development of BC. Although, the origin of HPV spreading to the mammary gland and its mechanism of dissemination is not clear. Methods: Seven serum samples from healthy donors and 58 from early BC patients collected pre-surgery were analyzed for the presence of HPV DNA. For 49/58 patients HPV DNA was analyzed also on the primary tumor tissue. 17 patients had luminal A BC (4 relapsed, 13 non relapsed), 16 had luminal B BC (5 relapsed, 11 non relapsed), 13 had triple-negative BC (6 relapsed, 7 non relapsed), 12 had HER2-positive BC (4 relapsed, 8 non relapsed). Circulating DNA was extracted from 500 μl of serum by Qiamp DNA Mini kit (Qiagen, Milan, Italy) and tumor DNA was extracted from at least four 10-micron sections by QIAamp DNA FFPE Tissue Kit (Qiagen, Milan, Italy). Circulating HPV DNA was amplified by a multiplex PCR with HPV E6 or E7 gene-specific primers and the sequence was assessed by a high-throughput MALDI-TOF mass spectrometry-based method. Results: HPV DNA was detected in only 5 serum of BC patients and in none of the healthy controls. 4/5 BC cases had high-risk HPV DNA (type 39,45,52,59) and 1 had type 73 low-risk HPV DNA. 4/5 HPV DNA-positive patients had previously low-grade cervical intraepithelial neoplasia (CIN I) detected by Pap smear. These 5 patients with circulating HPV DNA did not show HPV positivity in the BC tissue. 2 out of 49 cases were positive for universal HPV DNA sequence in tissue and only 1 case showed HPV type 51. No relation was found between HPV infection and tumor subtype or prognosis, neither for HPV DNA positivity between serum and tissue. Conclusions: Our data support the feasibility of HPV DNA detection by liquid biopsy in BC. The presence of circulating HPV could be due to a viral spread from other organs. More data are needed to establish the role of circulating HPV DNA and its potential association with HPV infection of the breast and/or of the cervix. [Table: see text]

Abstract 823: A case-control study of constitutional BRCA1 methylation in a mammographically screened cohort

Abstract Introduction: Over 90% of breast cancers occur sporadically with no obvious hereditary factors at play, therefore highlighting the need for biomarkers that can identify women who are predisposed to developing breast cancer. DNA methylation of the BRCA1 promoter region is detectable at low levels in the peripheral blood of some women. This phenomenon is referred to as constitutional BRCA1 methylation. Peripheral blood methylation of the BRCA1 gene has been identified as a predisposition factor to the development of BRCA1 methylated tumors. We performed a case-control study to assess peripheral blood BRCA1 methylation from healthy women, and from women with breast cancer. This study aimed to establish the frequency of peripheral blood methylation of the BRCA1 gene in women with and without breast cancer, to further our understanding of the role of constitutional BRCA1 methylation in breast cancer predisposition. Methods: Constitutional BRCA1 methylation was assessed in peripheral blood DNA of breast cancer patients and healthy women. Whole blood DNA was obtained from healthy women (n=327 controls) and women with breast cancer (n=300 cases) as part of the LifePool Project. Blood was collected on the day of mammography with BreastScreen Victoria. We used a probe-based droplet digital PCR (ddPCR) assay designed to quantify methylated and unmethylated alleles at the BRCA1 promoter region. Results: Previous research using less-sensitive methodologies has reported a BRCA1 methylation frequency of 2-4% in peripheral blood of healthy women. In this study, there was no significant difference in the observed BRCA1 methylation frequency between cases and controls (6.6% and 6.4% respectively). The discrepancy between our observed methylation frequencies and previously reported data can be attributed to the higher sensitivity of the ddPCR methodology. Nevertheless, the level of BRCA1 methylation in cases was significantly higher than the level of methylation observed in controls, consistent with previous studies. In addition, the frequency of BRCA1 methylation was higher in women diagnosed with breast cancer under the age of 50 compared to women diagnosed over 50 years old (average methylation frequency = 12.5% and 5.3% respectively). Conclusions: ddPCR methodology enabled accurate quantification of methylated BRCA1 alleles in peripheral blood DNA down to less than 0.1%. In this selected population of women undergoing mammography, there was no difference in the frequency of detectable BRCA1 methylation between cases and controls. Once age was considered, detectable BRCA1 methylation showed an association with age of onset. Tumours for the women with breast cancer will be retrieved and tested for BRCA1 methylation, to further examine the link between the methylation observed in peripheral blood DNA and development of a BRCA1-methylated tumor. Citation Format: Basant Ebaid, Hongdo Do, Jia Min Pang, Lisa Devereux, Alexander Dobrovic. A case-control study of constitutional BRCA1 methylation in a mammographically screened cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 823.

Abstract P2-04-01: Human papillomavirus (HPV) DNA detection in breast cancer by liquid biopsy: Something new on the horizon?

Abstract Background The presence of human papillomavirus (HPV) DNA in breast cancer (BC) tissues has been widely investigated in recent years. HPV DNA has been detected in ductal lavage fluids and in serum-derived extracellular vesicles of patients with benign or in situ breast lesions. However, there are no data attesting to its presence in liquid biopsies of different BC subtypes or to its impact on prognosis. Methods We analyzed a total of 72 serum samples for the presence of circulating HPV DNA, of which 20 were from luminal A BC (5 relapsed, 15 non relapsed), 17 from luminal B BC (5 relapsed, 12 non relapsed), 15 from triple-negative BC (6 relapsed and 9 non relapsed), 12 from HER2-positive BC (3 relapsed, 9 non relapsed) and 8 from healthy subjects. Circulating DNA was extracted and purified from 500 μl of serum by Qiamp DNA minikit (Qiagen, Milan, Italy) according to the manufacturer's instructions. HPV DNA was assessed by a high-throughput MALDI-TOF mass spectrometry-based method (Mass Array Platform, Agena Bioscience, Hamburg, Germany). The DNA target sequence was amplified by a multiplex PCR with HPV E6 or E7 gene-specific primers. A primer for primer extension annealing to the amplified product was extended at its 3′ terminal base for each HPV type. Results HPV DNA was detected in 5 BC patients but in none of the healthy controls. None of the serum samples analyzed showed HPV DNA types 16 or 18. Four of the 5 BC cases had high-risk HPV DNA (type 39,45,52,59) and one had low-risk HPV DNA (type 73). The 4 patients with high risk HPV DNA had low-grade cervical intraepithelial neoplasia (CIN 1) detected by Pap smear prior to the diagnosis of BC. No relation was found between HPV infection and tumor subtype or prognosis. Our in vitro studies also revealed the active release of HPV DNA into the extracellular vesicle compartment of cervical cancer cells. Table 1.Features of BC patients positive for circulating HPV DNA.Sample IDAge (years)GradingSubtypeDeathRelapseHPV type, riskCervical lesion17471Luminal A-No59, highCIN 139783Luminal B20/03/2015No45, highCIN 145462Luminal B-No39, highCIN 160583TN03/01/2011Yes73, lowHyperplasia63523HER2-positive-Yes52, highCIN 1TN, triple negative; CIN 1:low-grade cervical intraepithelial neoplasia Conclusions Our findings support the feasibility of HPV DNA evaluation by liquid biopsy in BC. They also suggest that circulating HPV DNA in BC patients might be of cervical tissue origin and that the presence of HPV DNA in BC may be a consequence of its spreading from virus-infected tissue such as that of the uterine cervix. Citation Format: Bravaccini S, Ravaioli S, Rocca A, Maltoni R, Cristalli C, Marasco E, De Carolis S, Cricca M, Bonafè M. Human papillomavirus (HPV) DNA detection in breast cancer by liquid biopsy: Something new on the horizon? [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-04-01.

Methylation of NBPF1 as a novel marker for the detection of plasma cell-free DNA of breast cancer patients

BackgroundRecent studies revealed that tumor-specific gene methylation can be detected in the circulating cell-free DNA (cfDNA) of cancer patients; therefore, methylated cfDNA is considered a promising biomarker. Human neuroblastoma breakpoint family member 1 (NBPF1) was originally identified in a neuroblastoma (NB) patient. The present study is the first to evaluate the presence of NBPF1 gene methylation in cfDNA in plasma of breast cancer patients. MethodsDifferentially methylated cfDNA was screened using bisulfite sequencing with a next-generation sequencer (BS-seq) among 25 breast cancer patients, 25 patients with a benign breast disease and 25 healthy female volunteers. Then, five specific methylation sites in the NBPF1 gene were verified in 11 breast cancer samples, and two further sites in the NBPF1 promoter were detected in 52 breast cancer patients (stages I-III), 31 patients with benign breast disease and 30 healthy controls by using methylation-specific PCR (MSP). Furthermore, the association between the methylation statuses of NBPF1 and the clinicopathological characteristics of breast cancer patients was analyzed. ResultsBS-seq demonstrated that the NBPF1 methylation levels in breast cancer patients were higher than those in patients with benign breast disease and healthy controls. The MSP results showed that the methylation rates of two sites in the NBPF1 promoter were 67.1% and 61.4% in breast cancer patients, 48.2% and 59.6% in patients with benign breast disease, and 40.9% and 48.1% in healthy controls, respectively. The methylation rates of one site were significantly different among the three groups (p < .05), with the highest rate in breast cancer patients. Moreover, there was no statistically significant correlation between the NBPF1 promoter methylation and the major clinicopathological features of the patients. ConclusionsThese results indicate that hypermethylation of the NBPF1 promoter occurs in a significant proportion of breast tumors and that NBPF1-methylated cfDNA may serve as a potential tumor marker for breast cancer.

High-sensitivity assay for monitoring ESR1 mutations in circulating cell-free DNA of breast cancer patients receiving endocrine therapy

Approximately 70% of breast cancers (BCs) express estrogen receptor alpha (ERα) and are treated with endocrine therapy. However, the effectiveness of this therapy is limited by innate or acquired resistance in approximately one-third of patients. Activating mutations in the ESR1 gene that encodes ERα promote critical resistance mechanisms. Here, we developed a high sensitivity approach based on enhanced-ice-COLD-PCR for detecting ESR1 mutations. The method produced an enrichment up to 100-fold and allowed the unambiguous detection of ESR1 mutations even when they consisted of only 0.01% of the total ESR1 allelic fraction. After COLD-PCR enrichment, methods based on next-generation sequencing or droplet-digital PCR were employed to detect and quantify ESR1 mutations. We applied the method to detect ESR1 mutations in circulating free DNA from the plasma of 56 patients with metastatic ER-positive BC. Fifteen of these patients were found to have ESR1 mutations at codons 536–538. This study demonstrates the utility of the enhanced-ice-COLD-PCR approach for simplifying and improving the detection of ESR1 tumor mutations in liquid biopsies. Because of its high sensitivity, the approach may potentially be applicable to patients with non-metastatic disease.

Evaluation of Aberrant Methylation of RASSF1A , BCL-XL , ITGA6 , TCF3 and SNAIL2 Genes in Peripheral Blood Leukocyte DNA in Breast Cancer Patients

Purpose: Nowadays, breast cancer is the most common cancer in women that caused by defects in the signaling mechanisms that control cell proliferation and apoptosis. Recent findings suggest that epigenetic alterations are the key factors in the development of breast cancer. Methylation changes occur within CpG islands of promoters and induce gene silencing. Abnormal methylation can be used as a potential biomarker for diagnosis of various diseases including cancer. In this study, methylation changes of RASSF1A, TCF3, BCL-XL, SNAIL2 and ITGA6 genes were assessment as epigenetic biomarkers of breast cancer. Methods: 70 breast cancer samples and 70 normal samples were selected and identified with different Clinical and pathological data, which might be related with methylation changes. Breast cancer patients and normal blood samples were collected, and DNA was extracted from white blood cells. DNA samples were digested using methylationsensitive restriction enzymes to identify methylated sites. Unlike hypomethylated positions, hypermethylated sites were not digested using these enzymes, thus replication occurs by PCR reaction. Results: RASSF1A and TCF3 (in some cases) were significantly hypermethylated in breast cancer cases (P 0.05) except RASSF1A gene ethylation changes that shown reverse correlation with age of patients (P<0.05). Conclusion: This study demonstrated that RASSF1A, ITGA6 and TCF3 genes methylation status were changed during breast cancer and they can be used as molecular biomarkers for breast cancer diagnosis.

Examination of methylation changes of VIM, CXCR4, DOK7, and SPDEF genes in peripheral blood DNA in breast cancer patients.

Studying whole blood DNA methylation as a risk marker has valuable applications in either diagnosis or staging of breast cancer. We investigated whole blood DNA methylation status of VIM, CXCR4, DOK7, and SPDEF genes in breast cancer patients in comparison to healthy control subjects. 60 patients with breast cancer and 40 healthy controls were examined. Genomic DNA isolated from peripheral blood and restriction enzyme polymerase chain reaction (REP) method was applied for analysis. Real-time PCR was used to confirm methylation status of the aforementioned genes and therefore to find out the methylation differences between normal and breast cancer subjects. Level of DOK7 promoter hypomethylation in normal and breast cancer samples was significant (P-value = 0.001). The study, also, showed that hypomethylation of VIM and CXCR4 genes are significant in patients compared with normal cases (P-value < 0.05). Furthermore, SPDEF promoter hypomethylation was not significantly differed between normal and breast cancer samples (P-value = 0.2). Hypermethylation of DOK7 gene in DNA from patients affected with breast cancer offers a biomarker for diagnosis of the breast cancer. This study indicates that methylation status of VIM and CXCR4 genes changes in breast cancer; so, they can be used as molecular biomarkers in breast cancer prognosis.

Abstract PL2: Cell free plasma tumor DNA in breast oncology

Abstract In the past decade, there has been great interest in the use of circulating cell-free DNA to help guide therapy decisions in clinical oncology. Recent work by our group and others suggests that circulating cell-free plasma tumor DNA offers advantages compared to tissue biopsies for mutation profiling. This concept of “liquid biopsy” may allow for a more global genomic picture of metastatic disease since blood serves as a reservoir for all metastatic sites. In addition, cell-free plasma tumor DNA can be measured serially and quantitatively, presenting the possibility of using circulating plasma tumor DNA as a biomarker to measure disease burden and response to therapies for both early and late stage disease. In this session, I will review historic and recent studies demonstrating the clinical potential of measuring cell-free plasma tumor DNA in breast cancer patients for early stage and metastatic disease. I will also outline future steps needed to translate liquid biopsies from research use to clinical practice, and recent work highlighting the first multi-institutional prospective clinical trial evaluating cell-free plasma tumor DNA in early stage breast cancer patients undergoing neoadjuvant chemotherapy. Citation Format: Park BH. Cell free plasma tumor DNA in breast oncology [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PL2.

New single nucleotide polymorphisms (SNPs) in homologous recombination repair genes detected by microarray analysis in Polish breast cancer patients.

Breast cancer is the most common cause of malignancy and mortality in women worldwide. This study aimed at localising homologous recombination repair (HR) genes and their chromosomal loci and correlating their nucleotide variants with susceptibility to breast cancer. In this study, authors analysed the association between single nucleotide polymorphisms (SNPs) in homologous recombination repair genes and the incidence of breast cancer in the population of Polish women. Blood samples from 94 breast cancer patients were analysed as test group. Individuals were recruited into the study at the Department of Oncological Surgery and Breast Diseases of the Institute of the Polish Mother's Memorial Hospital in Lodz, Poland. Healthy controls (n=500) were obtained from the Biobank Laboratory, Department of Molecular Biophysics, University of Lodz. Then, DNA of breast cancer patients was compared with one of the disease-free women. The test was supported by microarray analysis. Statistically significant correlations were identified between breast cancer and 3 not described previously SNPs of homologous recombination repair genes BRCA1 and BRCA2: rs59004709, rs4986852 and rs1799950. Further studies on larger groups are warranted to support the hypothesis of correlation between the abovementioned genetic variants and breast cancer risk.

Methylation of the SEPT9_v2 promoter as a novel marker for the detection of circulating tumor DNA in breast cancer patients.

The aim of the present study was to evaluate the promoter methylation status of SEPT9_v2 in breast cancer and to detect this methylated gene in circulating tumor DNA (ctDNA) in plasma. Bisulfite sequencing was performed with a next generation sequencer. Methylation of the SEPT9_v2 promoter was found in 67%(8/12) of breast cancer cell lines and 53% (10/19) of breast tumor tissue, but not in normal breast tissue (0/19). A clear inverse correlation was observed between the expression of SEPT9_v2 mRNA and the methylation index (MI) both in cell lines and breast cancer tissues. The MI of SEPT9_v2 was significantly higher in non-basal subtype of breast cancer (13.0%, n=84) than in basal subtype (3.0%, n=23) (P<0.0001). Methylated SEPT9_v2 ctDNA in plasma was detected in 11% (9/82) of primary breast cancer patients and 52% (26/50) of metastatic breast cancer patients, but not in the healthy controls (0/51). These results indicate that SEPT9_v2 promoter hypermethylation, which silences the expression of SEPT9_v2 mRNA, is observed in a significant proportion of breast tumors, and that methylated SEPT9_v2 may serve as a novel tumor marker for breast cancer.

Abstract 497: PIK3CA mutational status in circulating tumor cells (CTCs) and corresponding circulating tumor DNA in breast cancer patients

Abstract AIMS: Molecular characterization of Circulating Tumor Cells (CTCs) and analysis of circulating tumor DNA (ctDNA) in cancer patients holds promise as an extremely powerful and reliable non-invasive clinical tool for the individual molecular profiling of each patient in real time. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is implicated in human cancers, and somatic mutations in the p110α catalytic subunit of PI3K, are very frequent and play a crucial role in response to molecular targeted therapies in breast cancer. In this study, we analyzed PIK3CA hotspot mutations (1633 G&amp;gt;A, 3140 A&amp;gt;G) in CTCs and corresponding ctDNA of early and metastatic breast cancer patients. We also examined whether there is a correlation between the presence of PIK3CA mutations in CTCs and ctDNA. METHODS: We used our highly sensitive methodology for the detection of PIK3CA hotspot mutations in exons 9 (1633 G&amp;gt;A) and 20 (3140 A&amp;gt;G), based on a combination of allele-specific PCR, asymmetric rapid PCR and high resolution melting analysis (HRMA) (Markou et al, CCR 2014). We analyzed PIK3CA hotspot mutations in the EpCAM-positive CTCs fraction and the corresponding plasma samples of: i) a group of 19 patients with operable breast cancer, ii) a group of 40 breast cancer patients with verified metastasis and iii) 30 healthy female volunteers. All ctDNA samples (extracted from 2 ml plasma) were examined for their DNA quality; to verify DNA quality, primers specific for the wild type in exactly the same PIK3CA gene region for exon 9 were used to assess for hotspots mutations. The mutation status of PIK3CA gene in ctDNA samples was detected by the developed methodology exactly as previously described. All samples were analyzed in triplicate. RESULTS: The assay is highly sensitive as it can detect 0.05% of mutated dsDNA in the presence of 99.95% wtDNA for both exons (9 and 20) and highly specific (0/30 healthy donors). PIK3CA hotspot mutations were identified in ctDNA in 12/40 (30%) of metastasis-verified breast cancer patients and 4/19 (21%) of operable breast cancer patients. In metastasis-verified breast cancer patients, the concordance between EpCAM- positive CTCs and ctDNA for 1633 G&amp;gt;A mutation was 72.5%, whereas the corresponding concordance for the 3140 A&amp;gt;G mutation was 97.5%. In operable breast cancer patients, the concordance between EpCAM-positive CTCs and ctDNA for the 1633 G&amp;gt;A mutation was 80%, whereas the corresponding concordance for the 3140 A&amp;gt;G mutation was 100%. CONCLUSIONS: Detection of PIK3CA hotspot mutations in EpCAM-positive CTCs and corresponding ctDNA has shown a correlation both in metastasis-verified and operable breast cancer patients. We will further evaluate our findings in a large cohort of patients before and after treatment, to evaluate response to molecular targeted therapies in breast cancer. Citation Format: Elena Tzanikou, Athina Markou, Nikos Malamos, Vasilis Georgoulias, Evi S. Lianidou. PIK3CA mutational status in circulating tumor cells (CTCs) and corresponding circulating tumor DNA in breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 497.

Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients.

Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell-free DNA (cfDNA). Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n = 43), bone (n = 12) and brain metastases (n = 38), and cfDNA (n = 29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. ESR1 mutations were detected for D538G (n = 13), Y537S (n = 3), and Y537C (n = 1), and not for K303R, S463P, or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3% to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n = 5), mutations were detected in both (n = 3) or in cfDNA only (n = 2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1-mutant clones are enriched by endocrine therapy. Further studies should address whether sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. See related commentary by Gu and Fuqua, p. 1034.

Circulating Tumor DNA—the Potential of Liquid Biopsies

Recent studies suggest circulating cell-free tumor DNA offers advantages compared to tissue biopsies for mutation profiling. This concept of “liquid biopsy” allows for a more global genomic picture of metastatic disease since blood serves as a reservoir for all metastatic sites. In addition, cell-free tumor DNA can be measured quantitatively, presenting the possibility of using circulating tumor DNA as a biomarker to measure disease burden and response to therapies. Here, we review historic and recent studies demonstrating the clinical potential of measuring cell-free tumor DNA in breast cancer patients and future steps needed to translate liquid biopsies from research to clinical practice.

Promoter methylation of TRIM9 as a marker for detection of circulating tumor DNA in breast cancer patients.

The aim of the present study was to investigate the promoter methylation status of TRIM9 in breast cancer and to determine the presence of TRIM9-methylated circulating tumor DNA (ctDNA) in plasma. Bisulfite sequencing with a next generation sequencer showed TRIM9 promoter methylation in 92 % (11/12) of breast cancer cell lines (BCCs) and 68 % (13/19) of breast tumor tissues but not in any normal breast tissues (0/19). Methylation ratio of TRIM9 was significantly lower in basal type (9 %, n = 23) than luminal A (69 %, n = 29, P = 0.0003). Quantitative RT-PCR of BCCs disclosed an inverse correlation between TRIM9 mRNA expression and methylation ratio. TRIM9 methylated ctDNA in plasma was detected in 18 % (10/56) of metastatic breast cancer patients but not in any of 60 healthy controls. These results indicate that TRIM9 promoter hypermethylation, which suppresses TRIM9 mRNA expression, occurs in a significant proportion of breast tumors, and that TRIM9-methylated ctDNA thus may serve as a tumor marker for breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1423-7) contains supplementary material, which is available to authorized users.