Browsing by Author "Giannoni-Luza, S"

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Chip-based digital Polymerase Chain Reaction as quantitative technique for the detection of PIK3CA mutations in breast cancer patients
(Elsevier Ltd, 2022) Giannoni-Luza, S; Acosta, O; Murillo Carrasco, AG; Danos, P; Cotrina Concha, JM; Guerra Miller, H; Pinto, JA; Aguilar, A; Araujo, JM; Fujita, R; Buleje, J
Background: PIK3CA is a gene frequently mutated in breast cancer. With the FDA approval of alpelisib, the evaluation of PIK3CA for activating mutations is becoming routinely. Novel platforms for gene analysis as digital PCR (dPCR) are emerging as a potential replacement for the traditional Sanger sequencing. However, there are still few studies on chip-based dPCR to detect mutations in tumor samples. Thus, this cross-sectional study aimed to assess the sensibility of a chip-based dPCR to detect and quantify PIK3CA mutations and compare its performance with Sanger sequencing. Materials and Methods: Tumor samples from 57 breast cancer patients (22 pre-treatment samples, 32 tumors after neoadjuvant chemotherapy, and three lymph nodes) were collected and analyzed by Sanger sequencing and dPCR for the three PIK3CA most relevant mutations (p.E545K, p. H1047R, and p. H1047L). Digital PCR sensitivity, specificity, and overall performance were estimated by contingency tables, receptor operator characteristic (ROC), and area under the curve (AUC). Association of PIK3CA mutations with clinicopathological variables was conducted. Results: Sanger sequencing identified PIK3CA mutations in six patients (10.5%), two with p. H1047R, and four with p. E545K. Digital PCR confirmed those mutations and identified 19 additional patients with at least one mutation. Comparison between dPCR and Sanger sequencing showed a sensitivity of 100% (95% CI 53–100%), and a specificity of 84.2% (95% CI 83–84.2%). Besides, p. H1047R mutation detected by dPCR showed a significant association with breast cancer phenotype (p = 0.019) and lymphatic nodes infiltration (p = 0.046). Conclusions: Digital PCR showed a high sensitivity to detect mutations in tumor samples and it might be capable to detect low-rate mutations and tumor subpopulations not detected by Sanger sequencing. © 2022 The Author(s)
Promoter hypermethylation of RARB and GSTP1 genes in plasma cell-free DNA as breast cancer biomarkers in Peruvian women
(John Wiley and Sons Inc, 2023) Danos, P; Giannoni-Luza, S; Murillo-Carrasco, AG; Acosta, O; Guevara-Fujita, ML; Cotrina-Concha, JM; Guerra-Miller, H; Pinto-Oblitas, J; Aguilar-Cartagena, A; Araujo, JM; Fujita, R; Buleje-Sono, JL
Background: Promoter hypermethylation is one of the enabling mechanisms of hallmarks of cancer. Tumor suppressor genes like RARB and GSTP1 have been reported as hypermethylated in breast cancer tumors compared with normal tissues in several populations. This case–control study aimed to determine the association between the promoter methylation ratio (PMR) of RARB and GSTP1 genes (separately and as a group) with breast cancer and its clinical-pathological variables in Peruvian patients, using a liquid biopsy approach. Methods: A total of 58 breast cancer patients and 58 healthy controls, matched by age, participated in the study. We exacted cell-free DNA (cfDNA) from blood plasma and converted it by bisulfite salts. Methylight PCR was performed to obtain the PMR value of the studied genes. We determined the association between PMR and breast cancer, in addition to other clinicopathological variables. The sensitivity and specificity of the PMR of these genes were obtained. Results: A significant association was not found between breast cancer and the RARB PMR (OR = 1.90; 95% CI [0.62–6.18]; p = 0.210) or the GSTP1 PMR (OR = 6.57; 95% CI [0.75–307.66]; p = 0.114). The combination of the RARB + GSTP1 PMR was associated with breast cancer (OR = 2.81; 95% CI [1.02–8.22]; p = 0.026), controls under 50 years old (p = 0.048), patients older than 50 (p = 0.007), and postmenopausal (p = 0.034). The PMR of both genes showed a specificity of 86.21% and a sensitivity of 31.03%. Conclusion: Promoter hypermethylation of RARB + GSTP1 genes is associated with breast cancer, older age, and postmenopausal Peruvian patients. The methylated promoter of the RARB + GSTP1 genes needs further validation to be used as a biomarker for liquid biopsy and as a recommendation criterion for additional tests in asymptomatic women younger than 50 years.