hTERT gene methylation in circulating DNA, tumor, and surrounding tissue in breast cancer: a prospective study

ABSTRACT BACKGROUND: The human telomerase reverse transcriptase (hTERT) enzyme, encoded by the hTERT gene, synthesizes protective telomeric sequences on chromosomes and plays a fundamental role in cancer formation. Methylation of the hTERT gene has an upregulatory effect, increasing hTERT enzyme synthesis and allowing continuous tumor cell division. OBJECTIVE: In a group of patients with breast cancer, we aimed to analyze the methylation status of hTERT in the tumor, surrounding tissue, and circulating free deoxyribonucleic acid (cfDNA) of blood collected on the day of mastectomy and then approximately one year later. DESIGN AND SETTING: A prospective study was conducted at a university hospital in Rio de Janeiro, Brazil. METHODS: Samples were collected from 15 women with breast cancer on the day of mastectomy and approximately one year postoperatively. cfDNA was analyzed by sodium bisulfite conversion, followed by polymerase chain reaction, electrophoresis, and silver nitrate staining. RESULTS: Methylation of hTERT was detected in the tumors and surrounding tissues of all 15 patients. Five patients displayed hTERT methylation in the cfDNA from the blood of the first collection. Of the ten patients who returned for the second collection, three showed methylation. Two patients with methylation in the first collection did not display methylation in the second collection. One patient with no methylation in the first collection displayed methylation in the second collection, and one patient had a diminished level of methylation in the second collection. CONCLUSION: Only one-third of patients displayed methylation in their cfDNA, which may be related to the success of chemotherapy.


INTRODUCTION
Telomeres are repetitive deoxyribonucleic acid (DNA) sequences found at the ends of chromosomes that protect the chromosomes from degradation and ensure their stability.In each round of normal cell division, telomeres shorten, leading to cellular senescence and, on critical shortening, eventual cell death. 1,2Human telomerase reverse transcriptase(hTERT) is an enzyme that synthesizes telomeric DNA sequences. 3The hTERT enzyme is encoded by the hTERT gene and contains both a protein component and a ribonucleic acidcomponent. 2 The hTERT gene is not expressed in normal postnatal somatic cells, but its expression is elevated in stem, germ, and cancer cells. 4The expression of hTERT in cancer cells allows their continued division, maintains telomere length, and inactivates apoptosis.Various mechanisms enhance the regulation of hTERT in tumors, including amplifications, structural changes, mutations, and epigenetic changes. 5Epigenetic changes are DNA modifications, such as methylation, that do not alter the base sequence but may affect DNA expression.Methylation involves the addition of a methyl group to the cytosine base of cytosine-guanine dinucleotides. 5rculating cell-free DNA (cfDNA) is single or double-stranded extracellular DNA that is released into the bloodstream as a result of cell apoptosis, necrosis, and secretion. 6Increased levels of cfDNA and epigenetic changes are commonly observed in cancer patients and are associated with the progression of the disease and response to treatment. 7,8In cancer patients, cfDNA contains sequences from tumors which can serve as biomarkers for early-stage detection or for guiding therapy. 9,10A better understanding of cfDNA properties, in particular the methylation status, could guide future protocols for monitoring tumor composition and stage. 11In addition, cfDNA analysis allows cancer screening through liquid biopsy, 9,6 which has multiple advantages over tissue biopsies, such as not being limited to a single observational region, less invasiveness, and lower cost. 12

OBJECTIVE
This study aimed to investigate the methylation status of the hTERT gene in the tumors, surrounding tissues, and cfDNA of patients with breast cancer.Furthermore, the methylation status of hTERT in the cfDNA from blood collected on the day of mastectomy was compared with that from blood collected approximately one year later.To the best of our knowledge, this is the first study to track hTERT promoter methylation in cfDNA obtained at two different time points after breast cancer treatment.

Study design and patient recruitment
This prospective study included 15 women aged 44 to 78 years (mean age 56.7 ± 9.6 years) diagnosed with breast carcinoma.
Patients were admitted to the Institute of Gynecology, Universidade Federal do Rio de Janeiro, Brazil.All women underwent a total mastectomy as part of their treatment.Before surgery, patients were invited to voluntarily participate in the study, and those who agreed to participate signed a consent form after receiving all further clarifications.Recruitment took place between October 2018 and July 2021.The sample size was determined based on the availability of patients who met the study criteria during the recruitment period.

Data collection and ethical approval
Demographic and clinical data were obtained from the patients' medical records.This study was approved by the Clinical Research Ethics Committee of Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Brazil.Certificate: (CAAE) # 91406118.6.0000.5257,August 29, 2018.

Material collection
During mastectomy, sections measuring approximately 1 cm 3

Polymerase chain reaction (PCR)
To confirm the integrity of the DNA extracted from the samples, a fragment of exon 5 of the p53 gene was amplified by polymerase chain reaction (PCR).The amplification reaction was performed according to Pestaner et al., 14 generating a 274-base pair product.For the hTERT amplification, two pairs of primers were used as follows: hTERT-U(unmethylated) forward, 5′-GAGGTATTTCGGGAGGTTTCGC-3′ and hTERT-Ureverse, 5′-ACTCCGAACACCACGAATACCG-3′ producing a fragment of 126 base pairs, 15 and hTERT-M (methylated) forward, 5′-GGAGGTATTTTGGGAGGTTTTGT-3′and hTERT-M reverse, 5′CAAACTCCAAACACCACAAATACCA-3′ producing a fragment of 121 base pairs. 15PCR conditions were: initial denaturation at 96°C for 7 min followed by 35 cycles of 95°C for 1 min, 62°C for 1 min, and 72°C for 1 min.The final extension step was performed at 72°C for 5 min.

Gel electrophoresis and staining
PCR products were electrophoresed on 10% polyacrylamide gels.along with a negative control and a DNA marker.Gels were stained by the silver nitrate method. 16Briefly, the DNAcontaining gels were first fixed with ethanol and acetic acid, followed by impregnation with silver nitrate, and finally, incubation in sodium hydroxide and formaldehyde to reveal the DNA bands.

Methylation of the hTERT gene was detected in all 15 patients
in tumor cells and surrounding tissues (Figures 1A and B).
Two patients died after the first blood collection and three failed to return for the second blood collection for other reasons.
Of the ten patients who returned for the second blood collection, three (8, 10, and 14) displayed hTERT methylation (Figure 1D).expression is not observed in normal somatic cells.In contrast to other cancer-related genes, methylation of the hTERT promoter region does not lead to gene silencing, but instead has an upregulatory effect, increasing hTERT protein synthesis in tumor cells. 15For example, Haraguchi et al showed that hypermethylation of the hTERT gene in oral squamous cell carcinoma positively regulated hTERT enzyme synthesis, which was confirmed by immunohistochemistry. 15 According to the scientific literature, changes in DNA methylation are an early event in tumorigenesis. 10For instance; one study showed that changes in methylation were detected in plasma four years before clinical diagnosis. 18Our study investigated the hTERT gene methylation status in breast cancer patients.DNA was extracted from tumors, surrounding tissues, and peripheral blood (cfDNA).

Methylation of hTERT was measured in patients 3 and 13in the
The analyses revealed hTERT hypermethylation in tumors and surrounding tissues of all patients.This is very similar to the results reported by Masood et al., 19 who found that 94% of breast carcinomas displayed hypermethylation levels at least 2-fold higher than those measured in normal breast tissue.In contrast, in our study, only five of the fifteen patients presented hTERT methylation in their cfDNA.
This may be due to the efficacy of neoadjuvant chemotherapy before mastectomy; a successful treatment tends to considerably diminish the release of tumor cfDNA into the bloodstream.Furthermore, two patients (3 and 13) displayed methylation in the first blood collection but not in the second, which may also be indicative of the success of treatment before surgery.Similarly, patient 14 showed less methylation in the second collection than in the first, suggesting the effectiveness of neoadjuvant chemotherapy in reducing tumor cfDNA release into the bloodstream.

CONCLUSION
Our finding that only one-third of patients displayed methylation in their circulating DNA may be related to the success of chemotherapy.These results suggest that analysis of hTERT methylation in cfDNA could be useful in the follow-up of patients with breast cancer and in the evaluation of their response to treatment.
Further studies are required to analyze the methylation of other cancer-related genes in the cfDNA of patients with breast cancer.
were collected from the tumor and surrounding tissue of each patient for DNA analysis.In addition, 5 ml of peripheral blood was collected to analyze cfDNA.Another blood collection was made approximately one year later for the second cfDNA analysis.Sample collection and histopathological examination were performed at the Institute of Gynecology.Molecular analyses were performed at the Laboratório de Patologia Molecular, Hospital Universitário Clementino Fraga Filho.Circulating free DNA extraction DNA was extracted from fresh blood serum using a Quick-gDNA MiniPrep kit (Zymo Research, Orange County, United States), nº D3024, according to the manufacturer's standard protocol.Extraction of DNA from the tumor and surrounding tissue DNA extraction from the tumor and surrounding tissue was performed as previously described by McCormick et al., 13 using the Ultra Pure™ Phenol: Chloroform: Isoamyl Alcohol kit (Invitrogen™, Carlsbad, United States) Cat.No. 15593-031.Methylation mechanism DNA samples were modified with sodium bisulfite and analyzed using the Methylation-Specific Polymerase Chain Reaction method.DNA modification was performed using an EZ DNA Methylation-Gold TM Kit (Cat.No: D5005 Zymo Research, Orange County, United States), according to the standard protocol established by the manufacturer.

Table 2 .
Age and clinical data of patients TNM = tumor, node, metastasis; NC = neoadjuvant chemotherapy; WD = TNM stage not described in the records.