A Novel Detection Method of Breast Cancer through a Simple Panel of Biomarkers.

Circulating tumor cells (CTCs) have been identified as responsible for the spread of tumors to other organs of the body. In this sense, the development of sensitive and specific assays for their detection is important to reduce the number of deaths due to metastases. Here, we assessed whether the detection of CTCs in peripheral blood can serve in the construction of a panel of diagnosis and monitoring treatments of breast cancer (BC), focusing on the expression of markers of epithelial-mesenchymal transition. Through analyzing the blood from women without breast alterations (control), women with benign alterations, women with breast cancer without chemotherapy, and women with breast cancer with chemotherapy, we identified the best markers by transcriptional levels and determined three profiles of CTCs (mesenchymal, intermediate, and epithelial) by flow cytometry which, combined, can be used for diagnosis and therapy monitoring with sensitivity and specificity between 80% and 100%. Therefore, we have developed a method for detecting breast cancer based on the analysis of CTC profiles by epithelial-mesenchymal transition markers which, combined, can be used for the diagnosis and monitoring of therapy.

Raman spectroscopy of healthy, injured and amniotic membrane treated rat spinal cords.

Spinal cord injury is a significant public health issue with high psychological and financial costs to both the family and the society. Effective treatment strategies are hence of immense value. Several reports have suggested application of amniotic membrane for treating injuries, and there is evidence that it may be used to treat spinal injuries. In this animal model study, we explore biochemical changes in amniotic membrane treated injured spinal cord with respect to untreated injured and uninjured spinal cord using Raman spectroscopy. Multivariate statistical analysis is able to classify control, untreated, and treated with 92%, 87%, and 80% efficiency, respectively; suggesting unique biochemical changes in each group. Such studies may lead to development of minimally invasive methodologies for spinal cord injury treatment monitoring.

Association of baseline white blood cell counts with tuberculosis treatment outcome: a prospective multicentered cohort study.

OBJECTIVES: Tuberculosis (TB) is the leading infectious cause of death in the world. Cheaper and more accessible TB treatment monitoring methods are needed. Here, we evaluated white blood cell (WBC) absolute counts, lymphocyte, and monocyte proportions during TB treatment, and characterized their association with treatment failure. METHODS: This multicentered prospective cohort study was based in Bangladesh, Georgia, Lebanon, Madagascar, and Paraguay. Adult, non-immunocompromised patients with culture-confirmed pulmonary TB were included and followed up after two months of treatment and at the end of therapy. Blood counts were compared to treatment outcome using descriptive statistics, logistic regression, and Receiver Operating Characteristic (ROC) analyses. RESULTS: Between December 2017 and August 2020, 198 participants were enrolled, and 152 completed treatment, including 28 (18.5%) drug-resistant patients. The rate of cure at the end of treatment was 90.8% (138/152). WBC absolute counts decreased, and lymphocyte proportions increased throughout treatment. In multivariate analyses, baseline high WBC counts and low lymphocyte proportions were associated with positive sputum culture results at the end of treatment (WBC > 11,450 cells/mm3: p = 0.048; lymphocytes <16.0%: p = 0.039; WBC > 11,450 cells/mm3 and lymphocytes <16.0%: p = 0.024). CONCLUSION: High WBC counts and low lymphocyte proportions at baseline are significantly associated with the risk of TB treatment failure.

Assessing Parasite Load in Chagas Disease Patients by Quantitative Multiplex Real-Time PCR.

The development of accurate diagnostic tools and surrogate markers of parasitological response to treatment are priorities in Chagas disease (CD) research. For years, the detection of Trypanosoma cruzi DNA by PCR has proved to be useful in some clinical scenarios like acute CD, including cases of congenital transmission, CD reactivation in immunosuppressed patients, and posttreatment follow-up. In that sense, the implementation of quantitative real-time PCR (qPCR) assays was an important step in the development of more reliable tools for CD molecular diagnostics and treatment follow-up. In the last decade, two multicenter PCR studies allowed the harmonization and validation of standard operating procedures for PCR-based detection and quantification of T. cruzi DNA in blood samples. Herein we describe the two most used protocols to quantify parasitic load in human blood samples by multiplex qPCR assays and discuss some aspects to consider during planning and executing these procedures.

Usefulness of Serial Blood Sampling and PCR Replicates for Treatment Monitoring of Patients with Chronic Chagas Disease.

This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real-time quantitative PCR (qPCR) for baseline detection and quantification of parasitic loads and posttreatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely, DNDi-CH-E1224-001 (ClinicalTrials.gov registration no. NCT01489228) and the MSF-DNDi PCR Sampling Optimization Study (NCT01678599). Patients from Cochabamba (n = 294), Tarija (n = 257), and Aiquile (n = 220) were enrolled. Three serial blood samples were collected at each time point, and qPCR triplicates were tested for each sample. The first two samples were collected during the same day and the third one 7 days later. A patient was considered PCR positive if at least one qPCR replicate was detectable. Cumulative results of multiple samples and qPCR replicates enhanced the proportion of pretreatment sample positivity from 54.8% to 76.2%, 59.5% to 77.8%, and 73.5% to 90.2% in Cochabamba, Tarija, and Aiquile cohorts, respectively. This strategy increased the detection of treatment failure from 72.9% to 91.7%, 77.8% to 88.9%, and 42.9% to 69.1% for E1224 low-, short-, and high-dosage regimens, respectively, and from 4.6% to 15.9% and 9.5% to 32.1% for the benznidazole arm in the DNDi-CH-E1224-001 and MSF-DNDi studies, respectively. The addition of the third blood sample and third qPCR replicate in patients with nondetectable PCR results in the first two samples gave a small, non-statistically significant improvement in qPCR positivity. No change in clinical sensitivity was seen with a blood volume increase from 5 to 10 ml. The monitoring of patients treated with placebo in the DNDi-CH-E1224-001 trial revealed fluctuations in parasitic loads and occasionally nondetectable results. In conclusion, a serial sampling strategy enhanced PCR sensitivity to detecting treatment failure during follow-up and has the potential for improving recruitment capacity in Chagas disease trials, which require an initial positive qPCR result for patient admission.

Tendinopathy diagnosis and treatment monitoring using attenuated total reflectance-Fourier transform infrared spectroscopy.

Tendinopathy, an important sports injury afflicting athletes and general public, is associated with huge economic losses. The currently used diagnostic tests are subjective, show moderate sensitivity and specificity; while treatment failures persist despite advances in therapy. This highlights the need for tendinopathy diagnostic and treatment monitoring tools. This study investigates tendon injury, natural healing and effect of treatment using ATR-FTIR complemented with histopathology. Control (C), injured (I) and treated (T) rat tendons were extracted 3, 7, 14 and 28 days post-injury/treatment, representing phases of healing; and subjected to hematoxylin & eosin staining as well as spectroscopy. While C showed no change, I- and T-related histological changes could be clearly observed in stained sections. ATR-FTIR spectra highlighted the biochemical changes within groups. Multivariate analysis could classify C, I and T with 75%; different days between groups with 84%; and different days within group with 65% efficiency. Results suggest that such analysis can not only identify C, I or T but also different phases of healing. Difference between I and T at different time points also suggest change in rate of healing. Further studies may help develop this technique for clinical diagnosis and treatment monitoring in future.

Utility of urinary circulating tumor DNA for EGFR mutation detection in different stages of non-small cell lung cancer patients.

PURPOSE: Non-invasive methods of molecular profiling for non-small cell lung cancer (NSCLC) are useful for monitoring disease progression. The aim of the current study was to ascertain if transrenal DNA is sensitive for clinical correlation and EGFR detection in NSCLC patients. METHODS: 160 patients at various stages of the disease participated and samples were collected prospectively at 2-month intervals. A baseline sample was taken before treatment commencement. To ascertain the sensitivity of transrenal DNA, we compared its results with plasma DNA. ddPCR was used to profile the urine and blood samples for key EGFR mutations. RESULTS: Using tumor tissues as references, our study showed good concordance in EGFR mutations with transrenal DNA before treatment. Results were highly matching in late-stage NSCLC patients, with stage III/IV patients yielding an agreement of more than 90%. The assay was also sensitive to detect early-stage patients after surgical procedures. Profiles were highly concordant with results derived from plasma DNA, demonstrating the specificity of transrenal DNA assays. Serial monitoring of these patients showed stable molecular signatures and correlated to different treatments. Survival analysis showed good prognostic utility for late-stage patients with high transrenal DNA variations and patients that acquired T790M mutation. CONCLUSION: The study demonstrated the feasibility of using transrenal DNA in mutation profiling for different stages of NSCLC patients. It highlights the importance of continual monitoring and has potential clinical utility in the clinical management of NSCLC.