Outcome improvement with chemotherapy and radiotherapy in primary, localized, radiation-associated angiosarcoma of the breast region: a retrospective case series analysis.

BACKGROUND: We report on a series of consecutive patients with localized radiation-associated angiosarcoma (RAAS) of the breast region (BR) treated at two Italian sarcoma reference centers. MATERIALS AND METHODS: We retrospectively reviewed all cases of primary, localized, resectable RAAS of the BR, treated at one of the two participating institutions from 2000 to 2019. Relapse-free survival (RFS) and overall survival (OS) were calculated. The prognostic role of several variables was investigated. A propensity score matched (PSM) analysis was carried out. RESULTS: Eighty-four patients were retrospectively identified. Nineteen out of 84 patients (22.6%) were pretreated with an anthracycline-based regimen for previous cancer. All patients but one underwent surgery, with 37/84 (44.1%) receiving surgery alone and 46/84 (54.8%) a multimodal approach: 18/84 (21.4%) received radiation therapy (RT) and 46/84 (54.9%) received chemotherapy. An anthracycline-based regimen was used in 10/84 patients (11.9%), while a gemcitabine-based regimen was used in 33/84 (39.3%). With a median follow-up of 51 months (interquartile range: 30-126 months), 36/84 patients (42.9%) relapsed and 35/84 patients (41.7%) died (8/84, 9.5% in the lack of metastatic disease). Five-year OS and 5-year RFS were 57% [95% confidence interval (CI) 43% to 68%] and 52% (95% CI 39% to 63%), respectively. Both (neo)adjuvant RT and chemotherapy were associated with better RFS [hazard ratio (HR) 0.25, 95% CI 0.08-0.83; HR 0.45, 95% CI 0.23-0.89] with a trend towards a better OS (HR 0.51, 95% CI 0.18-1.46; HR 0.60, 95% CI 0.29-1.24). Gemcitabine-based regimens seemed to perform better (HR 4.28, 95% CI 1.29-14.14). PSM analysis retained the above results. CONCLUSIONS: This retrospective study supports the use of (neo)adjuvant RT and chemotherapy, in primary, localized resectable RAAS of the BR. An effort to prospectively validate the role of (neo)adjuvant RT and chemotherapy is warranted.

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.

Superior rectal artery preservation to reduce anastomotic leak rates in familial adenomatous polyposis patients treated with total colectomy and ileorectal anastomosis.

BACKGROUND: Total colectomy with ileorectal anastomosis (TC/IRA) is one of the prophylactic surgical options in patients with familial adenomatous polyposis (FAP). This study investigated the effectiveness of superior rectal artery (SRA) preservation during TC/IRA in reducing anastomotic leakage (AL). METHODS: This retrospective study was based on prospectively collected data (01/2000 - 12/2022) at the National Cancer Institute, Milan, Italy. FAP patients undergoing TC/IRA were enrolled. A 1:1  propensity score matching (PSM) was performed. Associations between SRA preservation and complications were investigated using univariate and multivariate analysis. RESULTS: The study population included 211 patients undergoing TC/IRA (Sex: 106 Male, 105 Female; Age: median 30 yrs, IQR: 20-48 yrs), 82 with SRA preservation (SRA group) and 129 without SRA preservation (controls). After PSM, 75 patients were considered for each group. SRA preservation was associated with fewer complications (OR 0.331, 95% CI 0.116; 0.942) in univariate logistic regression analysis. AL events were significantly fewer in the SRA group than in the control group (0 vs 12, p = 0.028). The SRA group had fewer overall surgical complication and pelvic sepsis rates (p = 0.020 and p = 0.028, respectively). Median operative time was significantly longer in the SRA group (340 min vs 240 min, p<0.001), and median hospital stay was significantly shorter (6 vs 7 days, p=0.017). Twenty-seven patients in the SRA group experienced intraoperative anastomotic bleeding, which was controlled endoscopically. Superimposable results were obtained analyzing the whole patient cohort. CONCLUSIONS: SRA preservation can be considered an advantage in this patient population, despite adding a further technical step during surgery and thereby prolonging the operative time. Intraoperative endoscopic checking of possible anastomotic bleeding sites is recommended.

A three-gene signature marks the time to locoregional recurrence in luminal-like breast cancer.

BACKGROUND: Gene expression profiling (GEP)-based prognostic signatures are being rapidly integrated into clinical decision making for systemic management of breast cancer patients. However, GEP remains relatively underdeveloped for locoregional risk assessment. Yet, locoregional recurrence (LRR), especially early after surgery, is associated with poor survival. PATIENTS AND METHODS: GEP was carried out on two independent luminal-like breast cancer cohorts of patients developing early (≤5 years after surgery) or late (>5 years) LRR and used, by a training and testing approach, to build a gene signature able to intercept women at risk of developing early LRR. The GEP data of two in silico datasets and of a third independent cohort were used to explore its prognostic value. RESULTS: Analysis of the first two cohorts led to the identification of three genes, CSTB, CCDC91 and ITGB1, whose expression, derived by principal component analysis, generated a three-gene signature significantly associated with early LRR in both cohorts (P value <0.001 and 0.005, respectively), overcoming the discriminatory capability of age, hormone receptor status and therapy. Remarkably, the integration of the signature with these clinical variables led to an area under the curve of 0.878 [95% confidence interval (CI) 0.810-0.945]. In in silico datasets we found that the three-gene signature retained its association, showing higher values in the early relapsed patients. Moreover, in the third additional cohort, the signature significantly associated with relapse-free survival (hazard ratio 1.56, 95% CI 1.04-2.35). CONCLUSIONS: Our three-gene signature represents a new exploitable tool to aid treatment choice in patients with luminal-like breast cancer at risk of developing early recurrence.

Skip pattern approach toward the early access of innovative anticancer drugs.

BACKGROUND: With the rapid development of innovative anticancer treatments, the optimization of tools able to accelerate the access of new drugs to the market by the regulatory authority is a major issue. The aim of the project was to propose a reliable methodological pathway for the assessment of clinical value of new therapeutic innovative options, to objectively identify drugs which deserve early access (EA) priority for solid and possibly in other cancer scenarios, such as the hematological ones. MATERIALS AND METHODS: After a comprehensive review of the European Public Assessment Report of 21 drugs, to which innovation had previously been attributed by the Italian Medicines Agency (Agenzia Italiana del Farmaco, AIFA), an expert panel formulated an algorithm for the balanced use of three parameters: Unmet Medical Need (UMN) according to AIFA criteria, Added Benefit (AB) according to the European Society for Medical Oncology's Magnitude of Clinical Benefit Scale (ESMO-MCBS) criteria and Quality of Evidence (QE) assessed by the Grades of Recommendation Assessment, Development and Evaluation (GRADE) method. By sequentially combining the above indicators, a final priority status (i.e. EA or not) was obtained using the skip pattern approach (SPA). RESULTS: By applying the SPA to the non-curative setting in solid cancers, the EA status was obtained by 5 out of 14 investigated drugs (36%); by enhancing the role of some categories of the UMN, additional 4 drugs, for a total of 9 (64%), reached the EA status: 2 and 3 drugs were excluded for not achieving an adequate score according to AB and QE criteria, respectively. For hematology cancer, only the UMN criteria were found to be adequate. CONCLUSIONS: The use of this model may represent a reliable tool for assessment available to the various stakeholders involved in the EA process and may help regulatory agencies in a more comprehensive and objective definition of new treatments' value in these contexts. Its generalizability in other national contexts needs further evaluation.

Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience.

BACKGROUND: DNA integrity is a critical part of the definition of genomic DNA (gDNA) quality and can influence downstream molecular applications. Pre-analytical variables as sample storage and DNA extraction methods can influence the quality and quantity of isolated DNA and affect molecular test performances. The aim of this paper is to investigate the role of blood sample storage and DNA extraction procedures on gDNA integrity and gDNA fragmentation impact on a molecular test. METHODS: 157 DNA samples deriving from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. 157 DNA samples derived from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), which aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. RESULTS/CONCLUSIONS: Our results demonstrate that blood sample storage and DNA extraction procedures influence gDNA integrity and that the same blood sample which underwent a long range multiplex PCR based analytical test can provide different results if the adopted pre-analytical procedures are not standardized.

Hepcidin and ferritin blood level as noninvasive tools for predicting breast cancer.

BACKGROUND: Currently used CA15-3 and CEA have found their clinical application particularly in the follow-up of patients with advanced disease. Novel biomarkers are urgent, especially for improving early diagnosis as well as for discriminating between benign and malignant disease. PATIENTS AND METHODS: In the present study, we used a proteomic approach based on surface-enhanced laser desorption/ionization-time of flight-mass spectrometry screening with the aim of identifying differentially expressed 2-30 kDa proteins in plasma of patients with malignant (65 cases) and benign (88 cases) breast lesions with respect to 121 healthy controls. RESULTS: We found that the most promising SELDI peaks were those corresponding to hepcidin-25 and ferritin light chain. We evaluated the capability of these peaks in predicting malignant and benign breast lesions using the area under the receiver operating characteristic curve (AUC). The results showed a good capacity to predict malignant breast lesions for hepcidin-25 [AUC: 0.82; 95% confidence interval (CI) 0.75-0.90] and ferritin light chain (AUC: 0.86; 95% CI 0.79-0.92). Conversely, a weak and satisfactory capability to predict benign breast lesion was observed for hepcidin-25 (AUC: 0.63; 95% CI 0.41-0.85) and ferritin light chain (AUC: 0.73; 95% CI 0.49-0.97). A significant association between HER2 status and hepcidin-25 was observed and the distribution of transferrin and ferritin were found significantly different in patients with breast cancer when compared with that of controls. CONCLUSIONS: This study provides evidence that hepcidin and ferritin light chain level in plasma may be of clinical usefulness to predict malignant and benign disease with respect to healthy controls.

SPIDIA-DNA: an External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses.

BACKGROUND: The EC-funded project SPIDIA is aimed to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for DNA molecular testing. To this purpose, a survey and a pan-European External Quality Assessment (EQA) were implemented. METHODS: SPIDIA facility sent to all the participants the same blood sample to be processed without time or temperature limitation. DNA quality parameters performed at SPIDIA facility included: UV spectrophotometric analysis of DNA purity and yield, PCR interferences study by Kineret software and DNA integrity analysis by pulsed field gel electrophoresis. RESULTS: 197 applications have been collected from 30 European countries. A high variability of DNA fragmentation was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A significant difference between the RNase P single copy gene quantity obtained in the DNA samples extracted with the precipitation-based method respect to those obtained with beads and column-based methods was observed. CONCLUSIONS: The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the pre-analytical phase of DNA analysis of blood samples.

SPIDIA-RNA: first external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

The diagnostic use of in vitro molecular assays can be limited by the lack of guidelines for collection, handling, stabilization and storage of patient specimens. One of the major goals of the EC funded project SPIDIA (www.spidia.eu) is to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for molecular testing which requires intracellular RNA analytes. To this end, a survey and a pan-European external quality assessment (EQA) were implemented. This report is the summary of the results of that trial. With the European Federation of Laboratory Medicine (EFLM) support, 124 applications for participation in the trial were received from 27 different European countries, and 102 laboratories actually participated in the trial. Each participating laboratory described their respective laboratory policies and practices as well as blood collection tubes typically used in performing this type of testing. The participating laboratories received two identical blood specimens: in an EDTA tubes (unstabilized blood; n=67) or in tubes designed specifically for the stabilization of intracellular RNA in blood (PAXgene® Blood RNA tubes; n=35). Laboratories were requested to perform RNA extraction according to the laboratory's own procedure as soon as possible upon receipt of the tubes for one tube and 24h after the first extraction for the second tube. Participants (n=93) returned the two extracted RNAs to SPIDIA facility for analysis, and provided details about the reagents and protocols they used for the extraction. At the SPIDIA facility responsible for coordinating the study, the survey data were classified, and the extracted RNA samples were evaluated for purity, yield, integrity, stability, and the presence of interfering substances affecting RT-qPCR assays. All participants received a report comparing the performance of the RNA they submitted to that of the other participants. All the results obtained by participants for each RNA quality parameter were classified as "in control", "warning", "out of control" and "missing" by consensus mean analysis. From the survey data, the most variable parameters were the volume of blood collected and the time and storage temperature between blood collection and RNA extraction. Analyzing the results of quality testing of submitted RNA samples we observed a data distribution of purity, yield, and presence of assay interference in agreement with expected values. The RNA Integrity Number (RIN) values distribution was, on the other hand, much wider than the optimal expected value, which led to an "in control" classification, even for partly degraded RNA samples. On the other hand, RIN values below 5 significantly correlated with a reduction of GAPDH expression levels. Furthermore, the distribution of the values of the four transcripts investigated (c-fos, IL-1ß, IL-8, and GAPDH) was wide and the RNA instability between samples separated by 24h were similar. Assuming the presence of at least two quality parameters "out of control" as an indication of a critical performance of the laboratory, 33% of the laboratories were included in this group. The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the pre-analytical phase of RNA analysis of blood samples.