Increased blood draws for ultrasensitive ctDNA and CTCs detection in early breast cancer patients.

Early breast cancer patients often experience relapse due to residual disease after treatment. Liquid biopsy is a methodology capable of detecting tumor components in blood, but low concentrations at early stages pose challenges. To detect them, next-generation sequencing has promise but entails complex processes. Exploring larger blood volumes could overcome detection limitations. Herein, a total of 282 high-volume plasma and blood-cell samples were collected for dual ctDNA/CTCs detection using a single droplet-digital PCR assay per patient. ctDNA and/or CTCs were detected in 100% of pre-treatment samples. On the other hand, post-treatment positive samples exhibited a minimum variant allele frequency of 0.003% for ctDNA and minimum cell number of 0.069 CTCs/mL of blood, surpassing previous investigations. Accurate prediction of residual disease before surgery was achieved in patients without a complete pathological response. A model utilizing ctDNA dynamics achieved an area under the ROC curve of 0.92 for predicting response. We detected disease recurrence in blood in the three patients who experienced a relapse, anticipating clinical relapse by 34.61, 9.10, and 7.59 months. This methodology provides an easily implemented alternative for ultrasensitive residual disease detection in early breast cancer patients.

Comparative study of droplet-digital PCR and absolute Q digital PCR for ctDNA detection in early-stage breast cancer patients.

BACKGROUND: Analysis of circulating tumor DNA (ctDNA) is increasingly used for clinical decision-making in oncology. However, ctDNA could represent ≤ 0.1 % of cell-free DNA in early-stage tumors and its detection requires high-sensitive techniques such as digital PCR (dPCR). METHODS: In 46 samples from patients with early-stage breast cancer, we compared two leading dPCR assays for ctDNA analysis: QX200 droplet digital PCR (ddPCR) system from Bio-Rad which is the gold-standard in the field, and Absolute Q plate-based digital PCR (pdPCR) system from Thermo Fisher Scientific which has not been reported before. We analyzed 5 mL of baseline plasma samples prior to any treatment. RESULTS: Both systems displayed a comparable sensitivity with no significant differences observed in mutant allele frequency. In fact, ddPCR and pdPCR possessed a concordance > 90 % in ctDNA positivity. Nevertheless, ddPCR exhibited higher variability and a longer workflow. Finally, we explored the association between ctDNA levels and clinicopathological features. Significantly higher ctDNA levels were present in patients with a Ki67 score > 20 % or with estrogen receptor-negative or triple-negative breast cancer subtypes. CONCLUSION: Both ddPCR and pdPCR may constitute sensitive and reliable tools for ctDNA analysis with an adequate agreement in early-stage breast cancer patients.

Development of a Novel NGS Methodology for Ultrasensitive Circulating Tumor DNA Detection as a Tool for Early-Stage Breast Cancer Diagnosis.

Breast cancer (BC) is the most prevalent cancer in women. While usually detected when localized, invasive procedures are still required for diagnosis. Herein, we developed a novel ultrasensitive pipeline to detect circulating tumor DNA (ctDNA) in a series of 75 plasma samples from localized BC patients prior to any medical intervention. We first performed a tumor-informed analysis to correlate the mutations found in tumor tissue and plasma. Disregarding the tumor data next, we developed an approach to detect tumor mutations in plasma. We observed a mutation concordance between the tumor and plasma of 29.50% with a sensitivity down to 0.03% in mutant variant allele frequency (VAF). We detected mutations in 33.78% of the samples, identifying eight patients with plasma-only mutations. Altogether, we determined a specificity of 86.36% and a positive predictive value of 88.46% for BC detection. We demonstrated an association between higher ctDNA median VAF and higher tumor grade, multiple plasma mutations with a likelihood of relapse and more frequent TP53 plasma mutations in hormone receptor-negative tumors. Overall, we have developed a unique ultra-sensitive sequencing workflow with a technology not previously employed in early BC, paving the way for its application in BC screening.

Aluminum Contamination in Parenteral Nutrition Admixtures for Low-Birth-Weight Preterm Infants in Mexico.

BACKGROUND: Aluminum contamination from intravenous solutions still represents an unsolved clinical and biochemical problem. Increased aluminum intake constitutes a risk factor for the development to metabolic bone disease, anemia, cholestasis, and neurocognitive alterations. Low-birth-weight preterm infants (LBWPIs) are one of the most exposed populations for aluminum toxicity. METHODS: To determine the presence of aluminum in components employed in the preparation of parenteral nutrition (PN) admixtures in Mexico and compare with the maximal aluminum recommended intake from the Food and Drug Administration. RESULTS: Cysteine, trace elements, levocarnitine, phosphate, and calcium salts tested positive for aluminum contamination. All components analyzed were contained in glass vials. Total aluminum intake for 2 sample PN admixtures were calculated in basis to cover nutrition requirements of 2 hypothetical LBWPIs. Aluminum contents, stratified in micrograms per kilogram of weight, exceeded maximal aluminum recommendations, particularly for the very LBWPIs. Substituting sodium phosphate for potassium phosphate salts reduced aluminum intake by 52.7%. Calcium gluconate was the leading aluminum contamination source and confers the greatest risk for aluminum overdose, even with the salt substitution of potassium phosphate by sodium phosphate salts. Adding cysteine and trace elements might increase aluminum content in PN admixtures. CONCLUSION: Cysteine, trace elements, phosphate, and gluconate salts are the main sources of aluminum in PN prepared in Mexico. Substituting sodium phosphate for potassium phosphate salts reduces aluminum intake but does not resolve aluminum contamination risk. Mineral salts contained in plastic vials should be explored as an additional measure to reduce aluminum contamination.