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

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.

Unlocking the complete blood count as a risk stratification tool for breast cancer using machine learning: a large scale retrospective study.

Optimizing early breast cancer (BC) detection requires effective risk assessment tools. This retrospective study from Brazil showcases the efficacy of machine learning in discerning complex patterns within routine blood tests, presenting a globally accessible and cost-effective approach for risk evaluation. We analyzed complete blood count (CBC) tests from 396,848 women aged 40-70, who underwent breast imaging or biopsies within six months after their CBC test. Of these, 2861 (0.72%) were identified as cases: 1882 with BC confirmed by anatomopathological tests, and 979 with highly suspicious imaging (BI-RADS 5). The remaining 393,987 participants (99.28%), with BI-RADS 1 or 2 results, were classified as controls. The database was divided into modeling (including training and validation) and testing sets based on diagnostic certainty. The testing set comprised cases confirmed by anatomopathology and controls cancer-free for 4.5-6.5 years post-CBC. Our ridge regression model, incorporating neutrophil-lymphocyte ratio, red blood cells, and age, achieved an AUC of 0.64 (95% CI 0.64-0.65). We also demonstrate that these results are slightly better than those from a boosting machine learning model, LightGBM, plus having the benefit of being fully interpretable. Using the probabilistic output from this model, we divided the study population into four risk groups: high, moderate, average, and low risk, which obtained relative ratios of BC of 1.99, 1.32, 1.02, and 0.42, respectively. The aim of this stratification was to streamline prioritization, potentially improving the early detection of breast cancer, particularly in resource-limited environments. As a risk stratification tool, this model offers the potential for personalized breast cancer screening by prioritizing women based on their individual risk, thereby indicating a shift from a broad population strategy.

Circulating tumor DNA: from discovery to clinical application in breast cancer.

Breast cancer (BC) stands out as the cancer with the highest incidence of morbidity and mortality among women worldwide, and its incidence rate is currently trending upwards. Improving the efficiency of breast cancer diagnosis and treatment is crucial, as it can effectively reduce the disease burden. Circulating tumor DNA (ctDNA) originates from the release of tumor cells and plays a pivotal role in the occurrence, development, and metastasis of breast cancer. In recent years, the widespread application of high-throughput analytical technology has made ctDNA a promising biomarker for early cancer detection, monitoring minimal residual disease, early recurrence monitoring, and predicting treatment outcomes. ctDNA-based approaches can effectively compensate for the shortcomings of traditional screening and monitoring methods, which fail to provide real-time information and prospective guidance for breast cancer diagnosis and treatment. This review summarizes the applications of ctDNA in various aspects of breast cancer, including screening, diagnosis, prognosis, treatment, and follow-up. It highlights the current research status in this field and emphasizes the potential for future large-scale clinical applications of ctDNA-based approaches.

Detection of circulating tumor cells by means of machine learning using Smart-Seq2 sequencing.

Circulating tumor cells (CTCs) are tumor cells that separate from the solid tumor and enter the bloodstream, which can cause metastasis. Detection and enumeration of CTCs show promising potential as a predictor for prognosis in cancer patients. Furthermore, single-cells sequencing is a technique that provides genetic information from individual cells and allows to classify them precisely and reliably. Sequencing data typically comprises thousands of gene expression reads per cell, which artificial intelligence algorithms can accurately analyze. This work presents machine-learning-based classifiers that differentiate CTCs from peripheral blood mononuclear cells (PBMCs) based on single cell RNA sequencing data. We developed four tree-based models and we trained and tested them on a dataset consisting of Smart-Seq2 sequenced data from primary tumor sections of breast cancer patients and PBMCs and on a public dataset with manually annotated CTC expression profiles from 34 metastatic breast patients, including triple-negative breast cancer. Our best models achieved about 95% balanced accuracy on the CTC test set on per cell basis, correctly detecting 133 out of 138 CTCs and CTC-PBMC clusters. Considering the non-invasive character of the liquid biopsy examination and our accurate results, we can conclude that our work has potential application value.

Association between red cell distribution width and all-cause mortality in patients with breast cancer: A retrospective analysis using MIMIC-IV 2.0.

PURPOSE: Investigating the association between red cell distribution width (RDW) and all-cause mortality in patients with breast cancer, to evaluate the potential clinical prognostic value of RDW. METHODS: Based on the RDW index, patients with breast cancer in the Medical Information Mart for Intensive Care (MIMIC-IV) database were categorized into quartiles. The primary outcomes included in-hospital mortality from all causes during the first six months, the first year, and the first three years. Cox hazards regression and restricted cubic spline (RCS) models were developed to investigate the effects of RDW on primary outcomes. RESULTS: The study included 939 patients (female). The 6-month, 1-year, and 3-year mortality rates were 14.0%, 21.4%, and 28.4%, respectively. Multivariate Cox proportional hazards analyses demonstrated that RDW exhibited an autonomous association with an increased risk of all-cause mortality. After adjusting for confounders, higher RDW quartiles were significantly associated with 6-month mortality (adjusted hazard ratio (HR), 3.197; 95% confidence interval (CI), 1.745-5.762; P < 0.001), 1-year mortality (adjusted HR, 2.978; 95% CI, 1.867-4.748; P < 0.001), and 3-year mortality (adjusted HR, 2.526; 95% CI, 1.701-3.750; P < 0.001). The RCS curves demonstrated that high RDW (> 14.6) was associated with a greater risk of all-cause mortality. Subgroup analyses revealed no statistically significant differences in the interactions between the subgroups. CONCLUSION: The study revealed a highly pronounced relationship between RDW and overall mortality, indicating its potential as an autonomous prognostic factor for increased mortality among patients with breast cancer.

Pleiotrophin serum level and metastasis occurrence in breast cancer patients.

BACKGROUND: Breast cancer (BC) cases in Makassar, Indonesia, are on the rise, with 2723 cases recorded in 2018. Tumor cells in the blood indicate metastasis, emphasizing the need for early diagnosis and monitoring. Pleiotrophin (PTN) is associated with various human malignancies, and recent studies suggest a correlation between PTN expression and advanced BC stages; therefore, PTN could serve as an independent predictor of metastasis. This study aimed to determine the correlation between serum PTN level, histopathological grading, and metastasis occurrence in BC patients in Makassar, Indonesia. METHODS: This study used an observational cross-sectional design. Pleiotrophin serum levels were examined using enzyme-linked immunosorbent assays. This study used a t-test and ROC curve analysis for the statistical tests. RESULTS: Of the 64 samples used in this study, metastasis was present in 26 cases and absent in 38 samples. The mean PTN serum levels in metastatic and non-metastatic breast cancer patients were 4.311 and 1.253, respectively. The PTN receiver operating characteristic curve showed an area under the curve of 2.47 ng/dL, which was statistically significant (p < 0.001). A significant relationship was found between PTN level and metastasis (p < 0.001). The correlation coefficient was 0.791, indicating a positive correlation. CONCLUSION: This study revealed that the serum PTN level among breast cancer patients had a cut-off value of 2.47 ng/dL. The research established a clear correlation between PTN level and metastasis occurrence in breast cancer patients, indicating a higher likelihood of distant metastasis with elevated PTN concentration.

Evaluation of some proinflammatory cytokines and biochemical parameters in pre and postmenopausal breast cancer women.

The study was planned to evaluate the differences in certain proinflammatory cytokines(IL-6, TNF-α) with CRP and biochemical parameters (E2, D3, LDH, GGT, TSB, Ca, Ph, uric acid), between women with pre- and postmenopausal breast cancer and seemingly healthy women in Iraqi women as controls; at medical city in teaching Oncology hospital,70 breast cancer patients women their ages ranged (47.51 ± 1.18) and 20 healthy women with age (44.45 ± 2.66) begun from September (2020) to February (2021). The aims of this study to investigate the evaluation of chemotherapy effects especially doxorubicin and cyclophosphamide only use in this study in pre and postmenopausal breast cancer women on proinflammatory cytokines(IL-6, TNF-α) with CRP and on biochemical parameters(E2, D3, LDH, GGT, TSB, Ca, Ph, uric acid) in pre and postmenapausal breast cancer women. The patients were divided into five groups and each group contains 14 patients women with breast cancer during pre and postmenopausal periods. The control groups were divided into 10 pre and 10 postmenopausal women(Fig. 1). The results of proinflammatory cytokines of and biochemical parameters in premenopausal groups were as the levels of IL-6 (pg/ml),TNF-α(pg/ml) and CRP (ng/ml) showed significant increase differences (P < 0.01)among breast cancer treated (BCT) groups in comparison with control groups,While the Liver enzymes GGT,LDH and TSB showed highly significant increase (P < 0.01) in BCT groups, Estrogen levels (pg/ml) and D3(ng/ml) increased significantly (P < 0.01)among BCT groups. Blood serum calcium and phosphorus with uric acid levels (mg/dl) showed significant difference (P < 0.01); While the result in postmenopausal of IL-6(pg/ml), TNF-α (pg/ml) and CRP (ng/ml) showed highly significant differences (P < 0.01)among BCT groups.While GGT(IU/L), LDH(IU/L) and TSB (mg/dl) enzymes were increased significantly (p < 0.01), Estrogen (pg/ml) and D3(ng/ml) levels showed significant increase (P < 0.01) among BCT groups.Blood calcium and phosphorus showed significant increase (P < 0.01) while uric acid was non-significant increase (P > 0.05).

Ultrasensitive and Rapid Circulating Tumor DNA Liquid Biopsy Using Surface-Confined Gene Amplification on Dispersible Magnetic Nano-Electrodes.

Circulating tumor DNA (ctDNA) detection has been acknowledged as a promising liquid biopsy approach for cancer diagnosis, with various ctDNA assays used for early detection and treatment monitoring. Dispersible magnetic nanoparticle-based electrochemical detection methods have been proposed as promising candidates for ctDNA detection based on the detection performance and features of the platform material. This study proposes a nanoparticle surface-localized genetic amplification approach by integrating Fe3O4-Au core-shell nanoparticles into polymerase chain reactions (PCR). These highly dispersible and magnetically responsive superparamagnetic nanoparticles act as nano-electrodes that amplify and accumulate target ctDNA in situ on the nanoparticle surface upon PCR amplification. These nanoparticles are subsequently captured and subjected to repetitive electrochemical measurements to induce reconfiguration-mediated signal amplification for ultrasensitive (∼3 aM) and rapid (∼7 min) metastatic breast cancer ctDNA detection in vitro. The detection platform can also detect metastatic biomarkers from in vivo samples, highlighting the potential for clinical applications and further expansion to rapid and ultrasensitive multiplex detection of various cancers.

Atypical memory B cells increase in the peripheral blood of patients with breast cancer regardless of lymph node involvement.

BACKGROUND: Breast cancer is the most common cancer in females. The immune system has a crucial role in the fight against cancer. B and T cells, the two main components of the adaptive immunity, are critical players that specifically target tumor cells. However, B cells, in contrast to T cells, and their role in cancer inhibition or progression is less investigated. Accordingly, in this study, we assessed and compared the frequency of naïve and different subsets of memory B cells in the peripheral blood of patients with breast cancer and healthy women. RESULTS: We found no significant differences in the frequencies of peripheral CD19+ B cells between the patients and controls. However, there was a significant decrease in the frequency of CD19+IgM+ B cells in patients compared to the control group (P=0.030). Moreover, the patients exhibited higher percentages of atypical memory B cells (CD19+CD27‒IgM‒, P=0.006) and a non-significant increasing trend in switched memory B cells (CD19+CD27+IgM‒, P=0.074). Further analysis revealed a higher frequency of atypical memory B cells (aMBCs) in the peripheral blood of patients without lymph node involvement as well as those with a tumor size greater than 2cm or with estrogen receptor (ER) negative/progesterone receptor (PR) negative tumors, compared with controls (P=0.030, P=0.040, P=0.031 and P=0.054, respectively). CONCLUSION: Atypical memory B cells (CD19+CD27‒IgM‒) showed a significant increase in the peripheral blood of patients with breast cancer compared to the control group. This increase seems to be associated with tumor characteristics. Nevertheless, additional research is necessary to determine the precise role of these cells during breast cancer progression.

Serial Postoperative Circulating Tumor DNA Assessment Has Strong Prognostic Value During Long-Term Follow-Up in Patients With Breast Cancer.

PURPOSE: Here, we report the sensitivity of a personalized, tumor-informed circulating tumor DNA (ctDNA) assay (Signatera) for detection of molecular relapse during long-term follow-up of patients with breast cancer. METHODS: A total of 156 patients with primary breast cancer were monitored clinically for up to 12 years after surgery and adjuvant chemotherapy. Semiannual blood samples were prospectively collected, and analyzed retrospectively to detect residual disease by ultradeep sequencing using ctDNA assays, developed from primary tumor whole-exome sequencing data. RESULTS: Personalized Signatera assays detected ctDNA ahead of clinical or radiologic relapse in 30 of the 34 patients who relapsed (patient-level sensitivity of 88.2%). Relapse was predicted with a lead interval of up to 38 months (median, 10.5 months; range, 0-38 months), and ctDNA positivity was associated with shorter relapse-free survival (P < .0001) and overall survival (P < .0001). All relapsing triple-negative patients (n = 7/23) had a ctDNA-positive test within a median of 8 months (range, 0-19 months), while the 16 nonrelapsed patients with triple-negative breast cancer remained ctDNA-negative during a median follow-up of 58 months (range, 8-99 months). The four patients who had negative tests before relapse all had hormone receptor-positive (HR+) disease and conversely, five of the 122 nonrelapsed patients (all HR+) had an occasional positive test. CONCLUSION: Serial postoperative ctDNA assessment has strong prognostic value, provides a potential window for earlier therapeutic intervention, and may enable more effective monitoring than current clinical tests such as cancer antigen 15-3. Our study provides evidence that those with serially negative ctDNA tests have superior clinical outcomes, providing reassurance to patients with breast cancer. For select cases with HR+ disease, decisions about treatment management might require serial monitoring despite the ctDNA-positive result.

Exploring the Immunological Profile in Breast Cancer: Recent Advances in Diagnosis and Prognosis through Circulating Tumor Cells.

This review offers a comprehensive exploration of the intricate immunological landscape of breast cancer (BC), focusing on recent advances in diagnosis and prognosis through the analysis of circulating tumor cells (CTCs). Positioned within the broader context of BC research, it underscores the pivotal role of the immune system in shaping the disease's progression. The primary objective of this investigation is to synthesize current knowledge on the immunological aspects of BC, with a particular emphasis on the diagnostic and prognostic potential offered by CTCs. This review adopts a thorough examination of the relevant literature, incorporating recent breakthroughs in the field. The methodology section succinctly outlines the approach, with a specific focus on CTC analysis and its implications for BC diagnosis and prognosis. Through this review, insights into the dynamic interplay between the immune system and BC are highlighted, with a specific emphasis on the role of CTCs in advancing diagnostic methodologies and refining prognostic assessments. Furthermore, this review presents objective and substantiated results, contributing to a deeper understanding of the immunological complexity in BC. In conclusion, this investigation underscores the significance of exploring the immunological profile of BC patients, providing valuable insights into novel advances in diagnosis and prognosis through the utilization of CTCs. The objective presentation of findings emphasizes the crucial role of the immune system in BC dynamics, thereby opening avenues for enhanced clinical management strategies.

Predictors of elevated C-reactive protein among pre-treatment, newly diagnosed breast cancer patients: A cross-sectional study.

BACKGROUND & AIMS: Accumulating evidence showed that inflammation contributes markedly to cancer progression, with C-reactive protein (CRP) being one of the lengthily studied inflammation marker. For breast cancer (BCa), pre-treatment elevated CRP upon diagnosis was linked with increased mortality. This study aimed to identify factors predictive of elevated CRP in pre-treatment BCa population that can serve as potential therapeutic targets to reduce inflammation. METHODS: This is a cross-sectional study using multiple logistic regression to identify predictors of elevated CRP among pre-treatment, newly diagnosed BCa patients. Studied variables were socio-demographic and medical characteristics, anthropometric measurements [body weight, Body Mass Index, body fat percentage, fat mass/fat free mass ratio, muscle mass, visceral fat], biochemical parameters [albumin, hemoglobin, white blood cell (WBC), neutrophil, lymphocyte], energy-adjusted Dietary Inflammatory Index, handgrip strength (HGS), scored Patient Generated-Subjective Global Assessment, physical activity level and perceived stress scale (PSS). RESULTS: A total of 105 participants took part in this study. Significant predictors of elevated CRP were body fat percentage (OR 1.222; 95 % CI 1.099-1.358; p < 0.001), PSS (OR 1.120; 95 % CI 1.026-1.223; p = 0.011), low vs normal HGS (OR 41.928; 95 % CI 2.155-815.728; p = 0.014), albumin (OR 0.779; 95 % CI 0.632-0.960; p = 0.019), and WBC (OR 1.418; 95% CI 1.024-1.963; p = 0.036). CONCLUSION: Overall, predictors of elevated CRP in pre-treatment, newly diagnosed BCa population were body fat percentage, PSS, HGS category, albumin and WBC.

The effect of tamoxifen on estradiol, SHBG, IGF-1, and CRP in women with breast cancer or at risk of developing breast cancer: a meta-analysis of randomized controlled trials.

BACKGROUND AND AIM: The effects of tamoxifen on the serum levels of hormones and acute phase reactants have been studied previously, but study results have been inconsistent, especially in women with breast cancer. Hence, we conducted this meta-analysis of randomized controlled trials (RCTs) to try to clarify the effects of tamoxifen on estradiol, insulin-like growth factor 1 (IGF-1), sex hormone binding globulin (SHBG), and C-reactive protein (CRP) serum levels in women with breast cancer or at risk of developing breast cancer. METHODS: Databases were systematically searched up to December 2023. The meta-analysis was generated through a random-effects model and is presented as the weighted mean difference (WMD) and 95 % confidence intervals (CI). RESULTS: Nine publications were included in the present meta-analysis. The comprehensive findings from the random-effects model revealed an elevation in estradiol (WMD: 13.04 pg/mL, 95 % CI: 0.79, 25.30, p = 0.037) and SHBG levels (WMD: 21.26 nmol/l, 95 % CI: 14.85, 27.68, p = 0.000), as well as a reduction in IGF-1 (WMD: -14.41 µg/L, 95 % CI: -24.23, -4.60, p = 0.004) and CRP concentrations (WMD: -1.17 mg/dL, 95 % CI: -2.29, -0.05, p = 0.039) following treatment with tamoxifen in women with breast cancer or at risk of developing breast cancer, with no impact on IGFBP-3 levels (WMD: 0.11 µg/mL, 95 % CI: -0.07, 0.30, p = 0.240). CONCLUSION: Tamoxifen administration seems to increase estradiol and SHBG levels and reduce CRP and IGF-1 levels in women with breast cancer or at risk of developing breast cancer. Further studies are needed to determine whether these changes have any clinical relevance.

CyTOF analysis revealed platelet heterogeneity in breast cancer patients received T-DM1 treatment.

T-DM1 (Trastuzumab Emtansine) belongs to class of Antibody-Drug Conjugates (ADC), where cytotoxic drugs are conjugated with the antibody Trastuzumab to specifically target HER2-positive cancer cells. Platelets, as vital components of the blood system, intricately influence the immune response to tumors through complex mechanisms. In our study, we examined platelet surface proteins in the plasma of patients before and after T-DM1 treatment, categorizing them based on treatment response. We identified a subgroup of platelets with elevated expression of CD63 and CD9 exclusively in patients with favorable treatment responses, while this subgroup was absent in patients with poor responses. Another noteworthy discovery was the elevated expression of CD36 in the platelet subgroups of patients exhibiting inadequate responses to treatment. These findings suggest that the expression of these platelet surface proteins may be correlated with the prognosis of T-DM1 treatment. These indicators offer valuable insights for predicting the therapeutic response to T-DM1 and may become important references in future clinical practice, contributing to a better understanding of the impact of ADC therapies and optimizing personalized cancer treatment strategies.

A highly sensitive and selective fluorescent biosensor for breast cancer derived exosomes using click reaction of azide-CD63 aptamer and alkyne-polymer dots.

Exosomes have gained recognition as valuable reservoirs of biomarkers, holding immense potential for early cancer detection. Consequently, there is a pressing need for the development of an economical and highly sensitive exosome detection methodology. In this work, we present a fluorescence method for breast cancer-derived exosome detection based on Cu-triggered click reaction of azide-modified CD63 aptamer and alkyne functionalized Pdots. The detection threshold for the exosomes obtained from the breast cancer serum was determined to be 6.09 × 107 particles per µL, while the measurable range spanned from 6.50 × 107 to 1.30 × 109 particles per µL. The employed methodology achieved notable success in accurately distinguishing breast cancer patients from healthy individuals through serum analysis. The application of this method showcases the significant potential for early exosome analysis in the clinical diagnosis of breast cancer patients.

Natural killer cell subsets and their functional molecules in peripheral blood of the patients with breast cancer.

BACKGROUND: Natural killer (NK) cells, CD3- lymphocytes, are critical players in cancer immune surveillance. This study aimed to assess two types of CD3- NK cell classifications (subsets), that is, convectional subsets (based on CD56 and CD16 expression) and new subsets (based on CD56, CD27, and CD11b expression), and their functional molecules in the peripheral blood of patients with breast cancer (BC) in comparison with healthy donors (HDs). METHODS: Thirty untreated females with BC and 20 age-matched healthy women were enrolled. Peripheral blood samples were collected and directly incubated with fluorochrome-conjugated antibodies against CD3, CD56, CD16, CD27, CD11b, CD96, NKG2C, NKG2D, NKp44, CXCR3, perforin, and granzyme B. Red blood cells were then lysed using lysing solution, and the stained cells were acquired on four-color flow cytometer. RESULT: Our results indicated 15% of lymphocytes in peripheral blood of patients with BC and HDs had NK cells phenotype. However, the frequency of total NK cells (CD3-CD56+), and NK subsets (based on conventional and new classifications) was not significantly different between patients and HDs. We observed mean fluorescent intensity (MFI) of CXCR3 in total NK cells (p = .02) and the conventional cytotoxic (CD3-CD56dim CD16+) NK cells (p = .03) were significantly elevated in the patients with BC compared to HDs. Despite this, the MFI of granzyme B expression in conventional regulatory (CD3-CD56brightCD16- /+) NK cells and CD3-CD56-CD16+ NK cells (p = .03 and p = .004, respectively) in the patients was lower than healthy subjects. CONCLUSION: The higher expression of chemokine receptor CXCR3 on total NK cells in patients with BC may be associated with increased chemotaxis-related NK cell infiltration. However, lower expression of granzyme B in conventional regulatory NK cells and CD3-CD56-CD16+ NK cells in the patients compared to HDs suggests reduced cytotoxic activity of the NK cells in BC. These results might demonstrate accumulating NK subsets with a dysfunctional phenotype in the peripheral blood of patients with BC.

Higher peripheral blood mitochondrial DNA copy number and relative telomere length in under 48 years Indonesian breast cancer patients.

OBJECTIVE: Breast cancer is the leading cause of cancer incidence and mortality among Indonesian women. A comprehensive investigation is required to enhance the early detection of this disease. Mitochondrial DNA copy number (mtDNA-CN) and relative telomere length (RTL) have been proposed as potential biomarkers for several cancer risks, as they are linked through oxidative stress mechanisms. We conducted a case-control study to examine peripheral blood mtDNA-CN and RTL patterns in Indonesian breast cancer patients (n = 175) and healthy individuals (n = 181). The relative ratios of mtDNA-CN and RTL were determined using quantitative real-time PCR (qPCR). RESULTS: Median values of mtDNA-CN and RTL were 1.62 and 0.70 in healthy subjects and 1.79 and 0.73 in breast cancer patients, respectively. We found a positive association between peripheral blood mtDNA-CN and RTL (p < 0.001). In under 48 years old breast cancer patients, higher peripheral blood mtDNA-CN (mtDNA-CN ≥ 1.73 (median), p = 0.009) and RTL (continuous variable, p = 0.010) were observed, compared to the corresponding healthy subjects. We also found a significantly higher 'High-High' pattern of mtDNA-CN and RTL in breast cancer patients under 48 years old (p = 0.011). Our findings suggest that peripheral blood mtDNA-CN and RTL could serve as additional minimally invasive biomarkers for breast cancer risk evaluation.

Mitochondrial Fraction of Circulating Cell-Free DNA as an Indicator of Human Pathology.

Circulating cell-free DNA (ccfDNA) of mitochondrial origin (ccf-mtDNA) consists of a minor fraction of total ccfDNA in blood or in other biological fluids. Aberrant levels of ccf-mtDNA have been observed in many pathologies. Here, we introduce a simple and effective standardized Taqman probe-based dual-qPCR assay for the simultaneous detection and relative quantification of nuclear and mitochondrial fragments of ccfDNA. Three pathologies of major burden, one malignancy (Breast Cancer, BrCa), one inflammatory (Osteoarthritis, OA) and one metabolic (Type 2 Diabetes, T2D), were studied. Higher levels of ccf-mtDNA were detected both in BrCa and T2D in relation to health, but not in OA. In BrCa, hormonal receptor status was associated with ccf-mtDNA levels. Machine learning analysis of ccf-mtDNA datasets was used to build biosignatures of clinical relevance. (A) a three-feature biosignature discriminating between health and BrCa (AUC: 0.887) and a five-feature biosignature for predicting the overall survival of BrCa patients (Concordance Index: 0.756). (B) a five-feature biosignature stratifying among T2D, prediabetes and health (AUC: 0.772); a five-feature biosignature discriminating between T2D and health (AUC: 0.797); and a four-feature biosignature identifying prediabetes from health (AUC: 0.795). (C) a biosignature including total plasma ccfDNA with very high performance in discriminating OA from health (AUC: 0.934). Aberrant ccf-mtDNA levels could have diagnostic/prognostic potential in BrCa and Diabetes, while the developed multiparameter biosignatures can add value to their clinical management.

Absolute lymphocyte count and neutrophil-to-lymphocyte ratio as predictors of CDK 4/6 inhibitor efficacy in advanced breast cancer.

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) are the standard agents for treating patients with estrogen receptor-positive and human epidermal growth factor receptor 2-negative advanced breast cancer (ER + HER2 - ABC). However, markers predicting the outcomes of CDK4/6i treatment have yet to be identified. This study was a single-center retrospective cohort study. We retrospectively evaluated 101 patients with ER + HER2 - ABC receiving CDK4/6i in combination with endocrine therapy at Fukuyama City Hospital between November 2017 and July 2021. We investigated the clinical outcomes and the safety of CDK4/6i treatment, and the absolute lymphocyte count (ALC) and neutrophil-to-lymphocyte ratio (NLR) as predictive markers for CDK4/6i. We defined the cut-off values as 1000/µL for ALC and 3 for NLR, and divided into "low" and "high" groups, respectively. We evaluated 43 and 58 patients who received abemaciclib and palbociclib, respectively. Patients with high ALC and low NLR had significantly longer overall survival than those with low ALC and high NLR (high vs. low; ALC: HR 0.29; 95% CI 0.12-0.70; NLR: HR 2.94; 95% CI 1.21-7.13). There was no significant difference in efficacy between abemaciclib and palbociclib and both had good safety profiles. We demonstrated that ALC and NLR might predict the outcomes of CDK4/6i treatment in patients with ER + HER2 - ABC.