Circulating microRNAs and therapy-associated cardiac events in HER2-positive breast cancer patients: an exploratory analysis from NeoALTTO.

PURPOSE: The relevance of cardiotoxicity in the context of HER2-positive breast cancer is likely to increase with increasing patient treatment exposure, number of treatment lines, and prolonged survival. Circulating biomarkers to early identify patients at risk of cardiotoxicity could allow personalized treatment and follow-up measures. The aim of this study is to examine the relationship between circulating microRNAs and adverse cardiac events in HER2-positive breast cancer patients. METHODS: We based our work on plasma samples from NeoALTTO trial obtained at baseline, after 2 weeks of anti-HER2 therapy, and immediately before surgery. Eleven patients experienced either a symptomatic or asymptomatic cardiac event. Circulating microRNAs were profiled in all patients presenting a cardiac event (case) and in an equal number of matched patients free of reported cardiac events (controls) using microRNA-Ready-to-Use PCR (Human panel I + II). Sensitivity analyses were performed by increasing the number of controls to 1:2 and 1:3. Normalized microRNA expression levels were compared between cases and controls using the non-parametric Kruskal-Wallis test. RESULTS: Eight circulating microRNAs resulted differentially expressed after 2 weeks of anti-HER2 therapy between patients experiencing or not a cardiac event. Specifically, the expression of miR-125b-5p, miR-409-3p, miR-15a-5p, miR-423-5p, miR-148a-3p, miR-99a-5p, and miR-320b increased in plasma of cases as compared to controls, while the expression of miR-642a-5p decreases. Functional enrichment analysis revealed that all these microRNAs were involved in cardiomyocyte adrenergic signaling pathway. CONCLUSION: This study provides proof of concept that circulating microRNAs tested soon after treatment start could serve as biomarkers of cardiotoxicity in a very early stage in breast cancer patients receiving anti-HER2 therapy.

Sarcopenic obesity and reduced BMD in young men living with HIV: body composition and sex steroids interplay.

PURPOSE: Sex steroids play a key role on male bone homeostasis and body composition (BC), their role in men living with HIV (MLWH) is less recognized. This study aimed at investigating the prevalence of low BMD, sarcopenia, and sarcopenic obesity (SO) and their relationship with sex steroids in MLWH aged < 50. METHODS: Prospective, cross-sectional, observational study on MLWH younger than 50 (median age 47.0 years). BC and BMD were evaluated with DXA. Two different definitions of sarcopenia were applied: appendicular lean mass/height2 (ALMI) < 7.26 kg/m2 or appendicular lean mass/body weight (ALM/W) < 28.27%. Low BMD was defined for Z-score < -2.0. Sarcopenia coupled with obesity identified SO. Serum total testosterone (T) and estradiol (E2) were measured by LC-MS/MS; free testosterone (cFT) was calculated by Vermeulen equation. RESULTS: Sarcopenia was detected in 107 (34.9%) and 44 (14.3%) out of 307 MLWH according to ALMI and ALM/W, respectively. The prevalence of SO was similar by using both ALMI (11.4%) and ALM/W (12.4%). Sarcopenic and SO MLWH had lower total T and cFT in both the definition for sarcopenia. BMD was reduced in 43/307 (14.0%). Serum E2 < 18 pg/mL was an independent contributing factor for sarcopenia, SO, and low BMD. CONCLUSIONS: T and E2 are important determinants of BC even in MLWH. This is among the first studies investigating the distribution of obesity phenotypes and the prevalence of SO among MLWH showing that SO is present in 11-12% of enrolled MLWH regardless of the definition used. However, deep differences emerged using two different diagnostic definitions.

The hypothalamic-pituitary-adrenal and -thyroid axes activation lasting one year after an earthquake swarm: results from a big data analysis.

PURPOSE: To cope physical and/or psychological threats, the human body activates multiple processes, mediated by a close interconnection among brain, endocrine and inflammatory systems. The aim of the study was to assess the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-thyroid (HPT) axes involvement after an acute stressful event (Emilia Romagna earthquake swarm) with a big data approach. METHODS: A retrospective, observational trial was performed, collecting all biochemical examinations regarding HPA and HPT axes performed in the same laboratory the year before and the year after the earthquake swarm (20-29 May 2012). RESULTS: Comparing 2576 pre-earthquake to 3021 post-earthquake measurements, a cortisol serum level increase was observed (p < 0.001). Similar increase was evident for urinary free cortisol (p = 0.016), but not for adrenocorticotropic hormone (p = 0.222). The biochemical hypercortisolism incidence increased from 7.6 to 10.3% after earthquakes (p = 0.001). Comparing 68,456 pre-earthquake to 116,521 post-earthquake measurements, a reduction in thyroid-stimulating hormone (TSH) levels was evident (p = 0.018), together with an increase in free triiodothyronine and free thyroxine levels (p < 0.001 and p < 0.001). Moreover, a significant increase in altered TSH after earthquakes was registered considering the epicenter-nearest measurements (p < 0.001). No clinically relevant alterations were observed considering thyroid-specific autoantibodies. CONCLUSION: A long-term HPA axis activation in the inhabitants of the earthquake-affected areas was highlighted for the first time. Moreover, an increased incidence of biochemical hypercortisolism emerged after earthquakes. We confirmed a recruitment of HPT axis after stressful events, together with increased incidence of altered TSH in the. Our big data study allowed to increase knowledge about the connection between external stressors and endocrine regulation.

The steroid response to human chorionic gonadotropin (hCG) stimulation in men with Klinefelter syndrome does not change using immunoassay or mass spectrometry.

PURPOSE: Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) was developed in parallel to Immunoassays (IAs) and today is proposed as the "gold standard" for steroid assays. Leydig cells of men with Klinefelter syndrome (KS) are able to respond to human chorionic gonadotropin (hCG) stimulation, even if testosterone (T) production was impaired. The aim was to evaluate how results obtained by IAs and LC-MS/MS can differently impact on the outcome of a clinical research on gonadal steroidogenesis after hCG stimulation. METHODS: A longitudinal, prospective, case-control clinical trial. (clinicaltrial.gov NCT02788136) was carried out, enrolling KS men and healthy age-matched controls, stimulated by hCG administration. Serum steroids were evaluated at baseline and for 5 days after intramuscular injection of 5000 IU hCG using both IAs and LC-MS/MS. RESULTS: 13 KS patients (36 ± 9 years) not receiving T replacement therapy and 14 controls (32 ± 8 years) were enrolled. T, progesterone, cortisol, 17-hydroxy-progesterone (17OHP) and androstenedione, were significantly higher using IAs than LC-MS/MS. IAs and LC-MS/MS showed direct correlation for all five steroids, although the constant overestimation detected by IAs. Either methodology found the same 17OHP and T increasing profile after hCG stimulation, with equal areas under the curves (AUCs). CONCLUSIONS: Although a linearity between IA and LC-MS/MS is demonstrated, LC-MS/MS is more sensitive and accurate, whereas IA shows a constant overestimation of sex steroid levels. This result suggests the need of reference intervals built on the specific assay. This fundamental difference between these two methodologies opens a deep reconsideration of what is needed to improve the accuracy of steroid hormone assays.

Early stage breast cancer follow-up in real-world clinical practice: the added value of cell free circulating tumor DNA.

PURPOSE: Physical examinations and annual mammography (minimal follow-up) are as effective as laboratory/imaging tests (intensive follow-up) in detecting breast cancer (BC) recurrence. This statement is now challenged by the availability of new diagnostic tools for asymptomatic cases. Herein, we analyzed current practices and circulating tumor DNA (ctDNA) in monitoring high-risk BC patients treated with curative intent in a comprehensive cancer center. PATIENTS AND METHODS: Forty-two consecutive triple negative BC patients undergoing neoadjuvant therapy and surgery were prospectively enrolled. Data from plasma samples and surveillance procedures were analyzed to report the diagnostic pattern of relapsed cases, i.e., by symptoms, follow-up procedures and ctDNA. RESULTS: Besides minimal follow-up, 97% and 79% of patients had at least 1 non-recommended imaging and laboratory tests for surveillance purposes. During a median follow-up of 5.1(IQR, 4.1-5.9) years, 13 events occurred (1 contralateral BC, 1 loco-regional recurrence, 10 metastases, and 1 death). Five recurrent cases were diagnosed by intensive follow-up, 5 by symptoms, and 2 incidentally. ctDNA antedated disseminated disease in all evaluable cases excepted two with bone-only and single liver metastases. The mean time from ctDNA detection to suspicious findings at follow-up imaging was 3.81(SD, 2.68), and to definitive recurrence diagnosis 8(SD, 2.98) months. ctDNA was undetectable in the absence of disease and in two suspected cases not subsequently confirmed. CONCLUSIONS: Some relapses are still symptomatic despite the extensive use of intensive follow-up. ctDNA is a specific test, sensitive enough to detect recurrence before other methods, suitable for clarifying equivocal imaging, and exploitable for salvage therapy in asymptomatic BC survivors.

Mammographic density to predict response to neoadjuvant systemic breast cancer therapy.

BACKGROUND: Mammographic density (MD) is a risk factor for breast cancer (BC) development, and recurrence. However, its predictive value has been less studied. Herein, we challenged MD as a biomarker associated with response in patients treated with neoadjuvant therapy (NAT). METHODS: Data on all NAT treated BC patients prospectively collected in the registry of Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy (2009-2019) were identified. Diagnostic mammograms were used to evaluate and score MD as categorized by the Breast Imaging-Reporting and Data System (BI-RADS), which identifies 4 levels of MD in keeping with relative increase of fibro-glandular over fat tissue. Each case was classified according to the following categories a (MD < 25%), b (26-50%), c (51-75%), and d (> 75%). The association between MD and pathological complete response (pCR), i.e., absence of BC cells in surgical specimens, was analyzed in multivariable setting used logistic regression models with adjustment for clinical and pathological variables. RESULTS: A total of 442 patients were analyzed, 120 of which (27.1%) attained a pCR. BI-RADS categories a, b, c, and d accounted for 10.0%, 37.8%, 37.1% and 15.2% of cases. Corresponding pCR were 20.5%, 26.9%, 30.5%, 23.9%, respectively. At multivariable analysis, when compared to cases classified as BI-RADS a, those with denser breast showed an increased likelihood of pCR with odds ratio (OR) of 1.70, 2.79, and 1.47 for b, c and d categories, respectively (p = 0.0996), independently of age, BMI [OR underweight versus (vs) normal = 3.76], clinical nodal and tumor status (OR T1/Tx vs T4 = 3.87), molecular subtype (HER2-positive vs luminal = 10.74; triple-negative vs luminal = 8.19). In subgroup analyses, the association of MD with pCR was remarkable in triple-negative (ORs of b, c and d versus a: 1.85, 2.49 and 1.55, respectively) and HER2-positive BC cases (ORs 2.70, 3.23, and 1.16). CONCLUSION: Patients with dense breast are more likely to attain a pCR at net of other predictive factors. The potential of MD to assist decisions on BC management and as a stratification factor in neoadjuvant clinical trials should be considered.

VMAT partial-breast irradiation: acute toxicity of hypofractionated schedules of 30 Gy in five daily fractions.

PURPOSE: To report acute toxicities in breast cancer (BC) patients (pts) recruited in a prospective trial and treated with accelerated partial-breast irradiation (APBI) using Volumetric Modulated Arc Therapy (VMAT) delivered with a hypofractionated schedule. METHODS: From March 2014 to June 2019, pts with early-stage BC (Stage I), who underwent breast conservative surgery (BCS), were recruited in a prospective study started at the National Cancer Institute of Milan. Pts received APBI with a hypofractionated schedule of 30 Gy in five daily fractions. Radiotherapy treatment (RT) was delivered using VMAT. Acute toxicity was assessed according to RTOG/EORTC criteria at the end of RT. RESULTS: Between March 2014 and June 2019, 151 pts were enrolled in this study. 79 Pts had right-side and 72 had left-side breast cancer. Median age was 69 (range 43-92). All pts presented with pathological stage IA BC, molecular classification was Luminal A in 128/151 (85%) and Luminal B in 23/151 (15%) cases. Acute toxicity, assessed at the end of RT, consisted of G1 erythema in 37/151 (24. 5%) pts and skin toxicities higher than G1, did not occur. Fibrosis G1 and G2 were reported in 41/151 (27. 1%) pts and in 2/151 pts (1. 3%), respectively. Edema G1 occurred in 8/151 (5. 3%) pts and asthenia G1 occurred in 1/151 (0. 6%) pts. CONCLUSIONS: APBI with VMAT proved to be feasible and can be a valid alternative treatment option after BCS in selected early breast cancer pts according to ASTRO guidelines. A longer follow-up is needed to assess late toxicity.

Axillary nodal involvement by primary tumor features in early breast cancer: an analysis of 2600 patients.

BACKGROUND: Primary tumor characteristics, which are readily available to all clinicians, may aid in selecting the optimal adjuvant therapy for patients with breast cancer (BC). Herein, we investigated the relationship between tumor size, hormone receptor and HER2 status, Ki67 and age with axillary lymph node metastases (ALNM) in early-BC patients. METHODS: We analyzed data on consecutive 2600 early-BC cases collected in the registry of Fondazione IRCC Istituto Nazionale dei Tumori, Milano, Italy. Correlation between Ki67 and primary tumor size (T-size) was calculated by Spearman's rank correlation coefficient. Association of ALNM with Ki67 and other tumor characteristics was investigated by logistic regression. Adjusted odds ratios (ORs) with 95% confidence intervals (95% CIs) were estimated in all cases, and separately analyzed according to age, T-size and BC subtype. RESULTS: Large tumor size strongly associated to ALNM, with an adjusted odds ratio (OR) for each 5-mm increase of 1.32 (95% CI 1.24-1.41), except for triple-negative BC (TNBC) cases. In tumors =10 mm, without lymphovascular invasion, representing the strongest predictor of ALNM (OR 6.09, 95% CI 4.93-7.53), Ki67 resulted particularly informative, with a fourfold increased odds of ALNM for values > 30%. CONCLUSIONS: These results raise the question whether axillary node status is redundant in cases with exceptionally good features, i.e., small tumors with low Ki67, or in those candidate to adjuvant systemic treatment/radiotherapy anyway including TNBC, and support the incorporation of primary BC tumor characteristics as stratification factors in ongoing trials aiming at de-escalating axillary surgical procedures.

Factors influencing acute and late toxicity in the era of adjuvant hypofractionated breast radiotherapy.

PURPOSE: To evaluate toxicity in breast cancer patients treated with anthracycline and taxane based chemotherapy and whole breast hypofractionated radiotherapy, and to identify the risk factors for toxicity. METHODS AND MATERIALS: 537 early breast cancer patients receiving hypofractionated radiotherapy after conservative surgery were enrolled from April 2009 to December 2014, in an Italian cancer institute. The dose was 42.4 Gy in 16 daily fractions, 2.65 Gy per fraction. The boost to the tumor bed was administered only in grade III breast cancer patients and in patients with close or positive margins. Acute and late toxicity were prospectively assessed during and after radiotherapy according to RTOG scale. The impact of patients clinical characteristics, performed treatments and dose inhomogeneities on the occurrence of an higher level of acute skin toxicity and late fibrosis has been evaluated by univariate and multivariate analysis. RESULTS: The mean age was 74 (range 46-91 yrs). 27% of patients received boost. 22% of cases (n = 119) received also chemotherapy. The median follow-up was 32 months. G1 and G2/G3 acute skin toxicity were 61.3% and 20.5% and G1 and G2/G3 late fibrosis 12.6% and 4.3% respectively. Chemotherapy (p = 0.04), diabetes (p = 0.04) and boost administration (p < 0.01) were found to be statistically significant on the occurrence of late fibrosis, but a multivariate analysis did not show any factors connected. The boost administration (p < 0.01), the breast volume (p = 0.05), dose inhomogeneities (p < 0.01) and boost volume (p = 0.04) were found to be statistically significant as concerns the occurrence of acute skin reaction at the univariate analysis, but only the boost administration (p = 0.02), at multivariate analysis. CONCLUSIONS: The results of our study, according to the large randomized trials, confirmed that hypofractionated whole breast irradiation is safe, and only the boost administration seems to be an important predictor for toxicity. Chemotherapy does not impact on acute and late skin toxicity.

Quality of clinical information: a new approach of EQA in Emilia-Romagna, Italy.

For correct enforcement of an external quality assessment (EQA) scheme, suitable parameters are required for the assessment of analytical performance. Traditional EQA schemes have always been chiefly concerned with the agreement of analytical results between laboratories. Although we consider this concept to be important, we also believe that particular attention must be paid to the quality of the clinical information, in relation to correct use of the results. On the basis of this principle, we have developed an EQA model which, besides considering the absolute value, also take the reference limits (RLs) into consideration by means of the normalization procedure. The evaluation of clinical information is of vital importance, especially in relation to immunoassays, since the low degree of standardization between methods, and the ensuing phenomenon of relative inaccuracy, make the use of suitable RLs essential. Actually, analysis of the results reveals a high degree of heterogeneity in the RLs used by the laboratories, even within the same method-groups.

MRI evaluation of neoadjuvant low-dose fractionated radiotherapy with concurrent chemotherapy in patients with locally advanced breast cancer.

OBJECTIVES: We address the diagnostic performance of breast MRI and the efficacy of neoadjuvant radiochemotherapy (NRC) treatment (NRC protocol) vs conventional neoadjuvant chemotherapy (NAC) in patients with locally advanced breast cancer. METHODS: The NRC protocol consists of six anthracycline/taxane cycles and concomitant low-dose radiotherapy on breast tumour volume. Breast MRI was performed at baseline and after the last therapy cycle in 18 and 36 patients undergoing the NRC protocol or conventional NAC (propensity matching). RESULTS: In both groups, we observed reduced tumour dimensions after the last cycle (p<0.001), and the response evaluation criteria in solid tumours (RECIST) class directly correlated with the tumour regression grade class after the last cycle (p<0.001). Patients in the NRC group displayed a higher frequency of complete/partial response than those in the NAC group (p=0.034). 17 out of 18 patients in the NRC group met the criteria for avoiding mastectomy based on final MRI evaluation. The RECIST classification displayed a superior diagnostic performance in the prediction of the response to treatment [area under the receiver operating characteristic curve (AUC)=0.72] than time-to-intensity curves and apparent diffusion coefficient (AUC 0.63 and 0.61). The association of the three above criteria yielded a better diagnostic performance, both in the general population (AUC=0.79) and in the NRC and the NAC group separately (AUC=0.82 and AUC=0.76). CONCLUSIONS: The pathological response is predicted by MRI performed after the last cycle, if both conventional MRI and diffusion imaging are integrated. The NRC treatment yields oncological results superior to NAC. Advances in knowledge MRI could be used to establish the neoadjuvant protocol in breast cancer patients.

Tumour markers in internal medicine: a low-cost test or an unnecessary expense? A retrospective study based on appropriateness.

OBJECTIVE AND BACKGROUND: In the last 35 years tumour markers (TM) have gained currency in clinical practice. However, in the light of indications by international guidelines, their use is often unjustified. Our aim was to quantify the use of some of the most common TM, assessing their appropriateness and their efficacy in an Internal Medicine Unit. METHODS: In the three Internal Medicine Units of the Department of Internal Medicine of Policlinico of Modena we have carried out a retrospective analysis of the assessment of the main TM (CEA, CA19.9, CA 125, CA 15.3, NSE). The analysis was divided into two distinct phases: (I) quantitative phase, in order to assess the scale of the problem in economical terms; (II) qualitative phase, in order to assess the efficacy of the tests and the appropriateness of their use. RESULTS: (I) At last one of the considered TM was requested in 5102 out of the 8253 admitted patients (62%) (period 2001-2003). The trend was similar in all three units examined. (II) The qualitative analyses revealed: (1) the most common motivation for their use (79%) was diagnostic, mostly prior to any other test; (2) a mere 5% of the requests were appropriate according to the international literature; and (3) TM showed a low positive predictive value when used for diagnosis in an unselected population such as that of an Internal Medicine unit. CONCLUSIONS: The results of our study showed that TM determination represents an overall cost for Internal Medicine units and that there is a high inappropriateness in their use compared to what it is suggested by international guidelines. Though the TM is a low-cost test when used correctly, it seems an unnecessary expense if not adequately incorporated into the decision making process.

Renal distal tubular handling of sodium in central fluid volume homoeostasis in preascitic cirrhosis.

BACKGROUND/AIMS: Patients with preascitic liver cirrhosis have an increased central plasma volume, and, for any given plasma aldosterone concentration, they excrete less sodium than healthy controls. A detailed study of the distribution of sodium reabsorption along the segments of the renal tubule, especially the distal one, is still lacking in preascitic cirrhosis. METHODS: Twelve patients with Child-Pugh class A cirrhosis and nine control subjects (both groups on a normosodic diet) were submitted to the following investigations: (a) plasma levels of active renin and aldosterone; (b) four hour renal clearance of lithium (an index of fluid delivery to the loop of Henle), creatinine, sodium, and potassium; (c) dopaminergic activity, as measured by incremental aldosterone response to intravenous metoclopramide. RESULTS: Metoclopramide induced higher incremental aldosterone responses, indicating increased dopaminergic activity in patients than controls, which is evidence of an increased central plasma volume (+30 min: 160.2 (68.8) v 83.6 (35.2) pg/ml, p<0.01; +60 min: 140.5 (80.3) v 36. 8 (36.1) pg/ml, p<0.01). Patients had increased distal fractional sodium reabsorption compared with controls (26.9 (6.7)% v 12.5 (3. 4)% of the filtered sodium load, p<0.05). In the patient group there was an inverse correlation between: (a) absolute distal sodium reabsorption and active renin (r -0.59, p<0.05); (b) fractional distal sodium reabsorption and sodium excretion (r -0.66, p<0.03). CONCLUSIONS: These data suggest that in preascitic cirrhosis the distal fractional tubular reabsorption of sodium is increased and critical in regulating both central fluid volume and sodium excretion.

Endogenous dopaminergic activity in Child-Pugh A cirrhosis: potential role in renal sodium handling and in the maintenance of clinical compensation.

BACKGROUND: We studied the main determinants of aldosterone secretion in a group of 20 patients with biopsy-proven Child-Pugh A cirrhosis without previous ascites or diuretic consumption. METHODS: We evaluated the plasma levels of adrenocorticotrophic hormone (ACTH), active renin and aldosterone (both supine at 07.00 h and after 30 min of upright posture),and active renin and aldosterone responses 30 min and 60 min after the administration of metoclopramide, a dopamine DA2 antagonist (10 mg e.v.). Nine normal subjects were also submitted to the metoclopramide stimulation test. RESULTS: Compared with control subjects, the patients showed significantly greater incremental aldosterone responses both 30 min and 60 min after metoclopramide (+30 min: 157.5+/-73.3 vs. 83.5+/-32.2 pg mL(-1), P< 0003; +60 min: 142.1+/-87.2 vs. 36.8+/-39.0 pg mL(-1), P < 0-001). We found significant positive correlations between amplitude of aldosterone response 30 min after metoclopramide and 24-h urinary fractional excretion of sodium (r=0.61, P < 0.01) and basal morning aldosterone levels (r=0.69, P < 0.001). CONCLUSIONS: The higher incremental aldosterone responses observed after metoclopramide in cirrhotic patients are expressions of increased dopaminergic activity in these patients compared with control subjects. Moreover, the correlation we found between the degree of dopaminergic activity and 24-h urinary fractional excretion of sodium suggests a role for endogenous dopamine as a relevant mediator of natriuresis in cirrhosis, at least in patients with compensated disease.