Rapid autopsies to enhance metastatic research: the UPTIDER post-mortem tissue donation program.

Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting.

HEBE project: Healthy aging versus inflamm-aging: The role of physical exercise in modulating the biomarkers of age-associated and environmentally determined chronic diseases, study protocol.

Inflamm-aging refers to the chronic low-grade inflammation that occurs with aging and cellular senescence, and it is linked to various diseases. Understanding the markers involved in inflammation and aging, as well as their interaction with environmental factors and bodily control mechanisms, can provide crucial tools for assessing the resilience (i.e. the ability to adapt and improve) of the human body, particularly in the presence of chronic degenerative conditions or vulnerable life stages, that place the individual and the community to which he belongs in a state of potential fragility. HEBE focuses on physical exercise, along with nutritional and lifestyle recommendations, to reduce systemic inflammation and promote healthy aging. HEBE encompasses multiple research lines (LR). In the ongoing LR1 ("proof of concept"), healthy lifestyle recommendations were provided to University of Milan employees, and changes in quality of life and well-being were assessed using a specialized questionnaire. The first 100 eligible subjects, who expressed their willingness to participate, underwent a personalized physical exercise protocol based on clinical and objective assessments. Biomedical samples were collected at baseline (T0) and follow-up (T1) to establish a shared biobank and identify non-invasive biomarkers that monitor the impact of physical exercise on individual characteristics such as cardiovascular and metabolic health. Subsequently (LR2-LR10), the proof of concept findings will be expanded to include various conditions of vulnerability such as obesity, cancer, endocrine disorders, cardiovascular diseases, infertility, functional syndromes, respiratory disorders, neurodegenerative diseases, and autoimmune conditions. The research lines will leverage the expertise of the 94 participating investigators to form a collaborative network that maximizes the potential for investigation and knowledge exchange. This approach fosters a culture of health promotion and disease prevention. ClinicalTrials.gov Identifier: NCT05815732.

Treatment Response, Tumor Infiltrating Lymphocytes and Clinical Outcomes in Inflammatory Breast Cancer-Treated with Neoadjuvant Systemic Therapy.

Inflammatory breast cancer (IBC) is a rare (1%-5%), aggressive form of breast cancer, accounting for approximately 10% of breast cancer mortality. In the localized setting, standard of care is neoadjuvant chemotherapy (NACT) ± anti-HER2 therapy, followed by surgery. Here we investigated associations between clinicopathologic variables, stromal tumor-infiltrating lymphocytes (sTIL), and pathologic complete response (pCR), and the prognostic value of pCR. We included 494 localized patients with IBC treated with NACT from October 1996 to October 2021 in eight European hospitals. Standard clinicopathologic variables were collected and central pathologic review was performed, including sTIL. Associations were assessed using Firth logistic regression models. Cox regressions were used to evaluate the role of pCR and residual cancer burden (RCB) on disease-free survival (DFS), distant recurrence-free survival (DRFS), and overall survival (OS). Distribution according to receptor status was as follows: 26.4% estrogen receptor negative (ER-)/HER2-; 22.0% ER-/HER2+; 37.4% ER+/HER2-, and 14.1% ER+/HER2+. Overall pCR rate was 26.3%, being highest in the HER2+ groups (45.9% for ER-/HER2+ and 42.9% for ER+/HER2+). sTILs were low (median: 5.3%), being highest in the ER-/HER2- group (median: 10%). High tumor grade, ER negativity, HER2 positivity, higher sTILs, and taxane-based NACT were significantly associated with pCR. pCR was associated with improved DFS, DRFS, and OS in multivariable analyses. RCB score in patients not achieving pCR was independently associated with survival. In conclusion, sTILs were low in IBC, but were predictive of pCR. Both pCR and RCB have an independent prognostic role in IBC treated with NACT. SIGNIFICANCE: IBC is a rare, but very aggressive type of breast cancer. The prognostic role of pCR after systemic therapy and the predictive value of sTILs for pCR are well established in the general breast cancer population; however, only limited information is available in IBC. We assembled the largest retrospective IBC series so far and demonstrated that sTIL is predictive of pCR. We emphasize that reaching pCR remains of utmost importance in IBC.

Histopathological growth patterns and tumor-infiltrating lymphocytes in breast cancer liver metastases.

Liver is the third most common organ for breast cancer (BC) metastasis. Two main histopathological growth patterns (HGP) exist in liver metastases (LM): desmoplastic and replacement. Although a reduced immunotherapy efficacy is reported in patients with LM, tumor-infiltrating lymphocytes (TIL) have not yet been investigated in BCLM. Here, we evaluate the distribution of the HGP and TIL in BCLM, and their association with clinicopathological variables and survival. We collect samples from surgically resected BCLM (n = 133 patients, 568 H&E sections) and post-mortem derived BCLM (n = 23 patients, 97 H&E sections). HGP is assessed as the proportion of tumor liver interface and categorized as pure-replacement ('pure r-HGP') or any-desmoplastic ('any d-HGP'). We score the TIL according to LM-specific guidelines. Associations with progression-free (PFS) and overall survival (OS) are assessed using Cox regressions. We observe a higher prevalence of 'any d-HGP' (56%) in the surgical samples and a higher prevalence of 'pure r-HGP' (83%) in the post-mortem samples. In the surgical cohort, no evidence of the association between HGP and clinicopathological characteristics is observed except with the laterality of the primary tumor (p value = 0.049) and the systemic preoperative treatment before liver surgery (p value = .039). TIL is less prevalent in 'pure r-HGP' as compared to 'any d-HGP' (p value = 0.001). 'Pure r-HGP' predicts worse PFS (HR: 2.65; CI: (1.45-4.82); p value = 0.001) and OS (HR: 3.10; CI: (1.29-7.46); p value = 0.011) in the multivariable analyses. To conclude, we demonstrate that BCLM with a 'pure r-HGP' is associated with less TIL and with the worse outcome when compared with BCLM with 'any d-HGP'. These findings suggest that HGP could be considered to refine treatment approaches.

Laparoscopic Cholecystectomy in Children: The Experience of Two Centers Focusing on Indications and Timing in the Era of "New Technologies".

BACKGROUND: In children, laparoscopic cholecystectomy (LC) is now considered the gold standard for gallbladder (GB) removal. In the past, hemolytic disorders associated with cholelithiasis represented the most frequent conditions requiring LC; this is being overtaken by cholelithiasis and biliary conditions in overweight or ex-premature children. AIMS: This study aims to describe current indications and timing for LC in pediatric patients. METHODS: Retrospective study. Data on previous medical therapy, ultrasound, pre- and intraoperative aspects, and histology were collected for patients treated in 2020-2023. RESULTS: In total, 45 patients were enrolled: 15 who underwent urgent surgery and 30 electives. Groups differed in terms of obesity rate, symptoms, ultrasound features, and intraoperative status. The most relevant risk factors for surgical complexity were age and pubertal stage, elevated cholestasis indexes, and gallbladder wall thickness > 3 mm at ultrasound. GB wall thickening ≥3 mm, US Murphy sign, fluid collections, and gallbladder distention on ultrasound correlated with high surgical scores. CONCLUSIONS: Indications for laparoscopic cholecystectomy in children seem to evolve caused by changing characteristics of the pediatric population. Patients with overweight/obesity may develop more complex GB diseases. Asymptomatic patients should be considered for surgery after observation, considering age and/or pubertal maturation when other risk factors are absent.

Environmental and Lifestyle Cancer Risk Factors: Shaping Extracellular Vesicle OncomiRs and Paving the Path to Cancer Development.

Intercellular communication has been transformed by the discovery of extracellular vesicles (EVs) and their cargo, including microRNAs (miRNAs), which play crucial roles in intercellular signaling. These EVs were previously disregarded as cellular debris but are now recognized as vital mediators of biological information transfer between cells. Furthermore, they respond not only to internal stimuli but also to environmental and lifestyle factors. Identifying EV-borne oncomiRs, a subset of miRNAs implicated in cancer development, could revolutionize our understanding of how environmental and lifestyle exposures contribute to oncogenesis. To investigate this, we studied the plasma levels of EV-borne oncomiRs in a population of 673 women and 238 men with a body mass index > 25 kg/m2 (SPHERE population). The top fifty oncomiRs associated with the three most common cancers in women (breast, colorectal, and lung carcinomas) and men (lung, prostate, and colorectal carcinomas) were selected from the OncomiR database. Only oncomiRs expressed in more than 20% of the population were considered for statistical analysis. Using a Multivariate Adaptive Regression Splines (MARS) model, we explored the interactions between environmental/lifestyle exposures and EV oncomiRs to develop optimized predictor combinations for each EV oncomiR. This innovative approach allowed us to better understand miRNA regulation in response to multiple environmental and lifestyle influences. By uncovering non-linear relationships among variables, we gained valuable insights into the complexity of miRNA regulatory networks. Ultimately, this research paves the way for comprehensive exposome studies in the future.

Association of body mass index with clinicopathological features and survival in patients with primary invasive lobular breast cancer.

PURPOSE: Invasive lobular carcinoma (ILC) represents up to 15% of all breast carcinomas. While the proportion of women with overweight and obesity increases globally, the impact of body mass index (BMI) at primary diagnosis on clinicopathological features of ILC and the prognosis of the patients has not been investigated yet. PATIENTS AND METHODS: We performed a multicentric retrospective study including patients diagnosed with non-metastatic pure ILC. The association of BMI at diagnosis with clinicopathological variables was assessed using linear or multinomial logistic regression. Univariable and multivariable survival analyses were performed to evaluate the association of BMI with disease-free survival (DFS), distant recurrence-free survival (DRFS), and overall survival (OS). RESULTS: The data of 2856 patients with ILC and available BMI at diagnosis were collected, of which 2570/2856 (90.0%) had oestrogen receptor (ER)-positive and human epidermal growth factor receptor (HER2) not amplified/overexpressed (ER+/HER2-) ILC. Of these 2570 patients, 80 were underweight (3.1%), 1410 were lean (54.9%), 712 were overweight (27.7%), and 368 were obese (14.3%). Older age at diagnosis, a higher tumour grade, a larger tumour size, a nodal involvement, and multifocality were associated with a higher BMI. In univariable models, higher BMI was associated with worse outcomes for all end-points (DFS: hazard ratio (HR) 1.21, 95CI 1.12-1.31, p value<0.01; DRFS: HR 1.25, 95CI 1.12-1.40, p value<0.01; OS: HR 1.25, 95CI 1.13-1.37, p value<0.01). This association was not statistically significant in multivariable analyses (DFS: HR 1.09, 95CI 0.99-1.20, p value 0.08; DRFS: HR 1.03, 95CI 0.89-1.20, p value 0.67; OS: HR 1.11, 95CI 0.99-1.24, p value 0.08), whereas grade, tumour size, and nodal involvement were still prognostic for all end-points. CONCLUSION: Worse prognostic factors such as higher grade, larger tumour size, and nodal involvement are associated with higher BMI in ER+/HER2- ILC, while there was no statistical evidence for an independent prognostic role for BMI. Therefore, we hypothesise that the effect of BMI on survival could be mediated through its association with these clinicopathological variables.

Association between metabolic syndrome, insulin resistance, and IGF-1 in breast cancer survivors of DIANA-5 study.

PURPOSE: Circulating insulin-like growth factor-1 (IGF-1) is positively associated with the risk of BC recurrence, and is more frequently dysregulated in older people, especially in those with metabolic syndrome (MetS) and obesity. This study aimed to analyze the association between IGF-1 levels and indices of MetS and insulin resistance in BC survivors. METHODS: Baseline data of 563 BC survivors enrolled in the DIet and ANdrogen-5 (DIANA-5; NCT05019989) study were analyzed. RESULTS: Lower circulating IGF-1 levels in subjects with MetS than in those without MetS were found. After stratification of the patients according to the diagnosis of MetS, we highlighted that the insulin was the main predictor of elevated IGF-1 levels only in subjects without MetS. Moreover, we found an interaction between high-density lipoprotein cholesterol (HDL-C), glycemia, and IGF-1 levels, showing a positive correlation between HDL-C and IGF-1, especially in subjects with higher values of glycemia and without a diagnosis of MetS. CONCLUSIONS: While IGF-1 levels appear to be much more impaired in subjects diagnosed with MetS, in non-MetS subjects, IGF-1 levels may respond better to metabolic parameters and lifestyle changes. Further studies are needed to analyze the role of physical activity and/or dietary intervention in modulating IGF-1 concentrations in BC survivors. IMPLICATIONS FOR CANCER SURVIVORS: These results could have important clinical implications for planning customized strategies aimed at modulating IGF-1 levels in BC survivors. In fact, while the IGF-1 system seems to be much more compromised in subjects with a diagnosis of MetS, in noMetS subjects, IGF-1 levels could better respond to lifestyle changes.

Individual risk prediction of high grade prostate cancer based on the combination between total prostate-specific antigen (PSA) and free to total PSA ratio.

OBJECTIVES: Clinical practice guidelines endorse the stratification of prostate cancer (PCa) risk according to individual total prostate-specific antigen (tPSA) values and age to enhance the individual risk-benefit ratio. We defined two nomograms to predict the individual risk of high and low grade PCa by combining the assay of tPSA and %free/tPSA (%f/tPSA) in patients with a pre-biopsy tPSA between 2 and 10 µg/L. METHODS: The study cohort consisted of 662 patients that had fPSA, tPSA, and a biopsy performed (41.3% with a final diagnosis of PCa). Logistic regression including age, tPSA and %f/tPSA was used to model the probability of having high or low grade cancer by defining 3 outcome levels: no PCa, low grade (International Society of Urological Pathology grade, ISUP<3) and high grade PCa (ISUP≥3). RESULTS: The nomogram identifying patients with: (a) high vs. those with low grade PCa and without the disease showed a good discriminating capability (∼80%), but the calibration showed a risk of underestimation for predictive probabilities >30% (a considerable critical threshold of risk), (b) ISUP<3 vs. those without the disease showed a discriminating capability of 63% and overestimates predictive probabilities >50%. In ISUP 5 a possible loss of PSA immunoreactivity has been observed. CONCLUSIONS: The estimated risk of high or low grade PCa by the nomograms may be of aid in the decision-making process, in particular in the case of critical comorbidities and when the digital rectal examinations are inconclusive. The improved characterization of the risk of ISUP≥3 might enhance the use for magnetic resonance imaging in this setting.

Comparison of the genomic alterations present in tumor samples from patients with metastatic inflammatory versus non-inflammatory breast cancer reveals AURKA as a potential treatment target.

Inflammatory breast cancer (IBC) is a rare but aggressive subtype of breast cancer, mainly characterized using primary tumor samples. Here, using public datasets, we compared the genomic alterations in primary and metastatic samples from patients with metastatic IBC versus patients with metastatic non-IBC. We observed a higher frequency of AURKA amplification in IBC. We further showed that AURKA amplification was associated with increased AURKA mRNA expression, which we demonstrated was higher in IBC. Finally, higher protein expression of AURKA was associated with worse prognosis in patients with IBC. These findings deserve further investigation given the existence of AURKA-inhibitors.

Managing the impact of inter-method bias of prostate specific antigen assays on biopsy referral: the key to move towards precision health in prostate cancer management.

OBJECTIVES: We assessed the inter-method bias of total (tPSA) and free (fPSA) prostate-specific antigen (PSA) immunoassays to establish if tPSA-based risk thresholds for advanced prostate cancer (PCa), obtained from one method (Roche) can be converted into the corresponding concentrations assayed by other methods. Then we evaluated the impact of the bias of tPSA and fPSA on the estimation of the %f/tPSA ratio and performed a re-calibration of the proposed thresholds for the %f/tPSA ratio according to the assay used. METHODS: tPSA and fPSA were measured in 135 and 137 serum samples, respectively by Abbott Alinity i, Beckman Access Dxl, Roche Cobas e801, and Siemens Atellica IM analytical platforms. Scatterplots, Bland-Altman diagrams, Passing-Bablok (PB) were used to inspect and estimate the systematic and proportional bias between the methods. The linear equations with confidence intervals of the parameter estimates were used to transform the tPSA risk thresholds for advanced PCa into the corresponding concentrations measurable by the other analytical methods. To construct a correction coefficient for converting the %f/tPSA ratio from one method to the other, PB and non-parametric boostrapping were used. RESULTS: The inter-method bias is not constant but strictly linear allowing the conversion of PSA results obtained from Roche into the other assays, which underestimate tPSA vs. Roche. Siemens and Abbott vs. Roche and Beckman assays, being characterized by a positive and a negative proportional bias for tPSA and fPSA measurements, tend to overestimate the %f/tPSA ratio. CONCLUSIONS: There is a consistent risk to miss advanced PCa, if appropriate conversion factors are not applied.

The association between adiposity and anti-proliferative response to neoadjuvant endocrine therapy with letrozole in post-menopausal patients with estrogen receptor positive breast cancer.

The impact of adiposity on the efficacy of endocrine treatment in patients with estrogen receptor positive breast cancer is poorly investigated. Here, we retrospectively investigated in a cohort of 56 patients whether body mass index and/or mammary adiposity are associated with anti-proliferative response in the neoadjuvant setting. Anti-proliferative response was defined as high Ki67 at baseline (Ki67bl) and low Ki67 at surgery (Ki67srg), using the 14% cut-off. Mammary adipocyte size was assessed on hematoxylin and eosin slides from the surgical samples using digital pathology. A higher proportion of tumors with an anti-proliferative response was observed in patients with obesity (54.5%) as compared to patients with normal weight (9.0%) and patients with overweight (40.0%) (p = 0.031), confirmed by multivariable regression analysis adjusted for baseline Ki67 (OR, obese vs normal weight: 13.76, 95%CI: 1.49-207.63, p = 0.020). Larger adipocyte diameter was identified as predictor of anti-proliferative response (OR per increase in diameter of 5 µm for adipocytes distant from the tumor: 2.24, 95%CI: 1.01-14.32, p = 0.046). This study suggests that anti-proliferative response to neoadjuvant letrozole might be more frequent in patients with increased systemic or mammary adiposity.

Timing evolution of lobular breast cancer through phylogenetic analysis.

BACKGROUND: Late distant recurrence is a challenge for the treatment of invasive lobular carcinoma (ILC) of the breast. Despite in-depth characterisation of primary ILC, the molecular landscape of metastatic ILC is still only partially understood. METHODS: We retrospectively identified 38 ILC patients from the tissue banks of six European institutions. DNA extracted from patient matched primary and metastatic FFPE tissue blocks was whole genome sequenced to compute somatic copy number aberrations. This, in turn, was used to infer the evolutionary history of these patients. FINDINGS: The data show different metastatic seeding patterns, with both an early and late divergence of the metastatic lineage observed in ILC. Additionally, cascading dissemination from a metastatic precursor was a dominant rule. Alterations in key cancer driver genes, such as TP53 or CCND1, were acquired early while additional aberrations were present only in the metastatic branch. In about 30% of the patients, the metastatic lineage harboured less aberrations than the primary tumour suggesting a period of tumour dormancy or prolonged adaptation at the distant site. This phenomenon was mostly observed in tumours from de novo metastatic patients. INTERPRETATION: Our results provide insights into ILC evolution and offer potential paths for optimised ILC care. FUNDING: This work has received financial support from Les Amis de l'Institut Bordet, MEDIC, the Breast Cancer Research Foundation (BCRF) and the Belgian Fonds National de la Recherche Scientifique (F.R.S-FNRS).

A Simulation Study to Compare the Predictive Performance of Survival Neural Networks with Cox Models for Clinical Trial Data.

BACKGROUND: Studies focusing on prediction models are widespread in medicine. There is a trend in applying machine learning (ML) by medical researchers and clinicians. Over the years, multiple ML algorithms have been adapted to censored data. However, the choice of methodology should be motivated by the real-life data and their complexity. Here, the predictive performance of ML techniques is compared with statistical models in a simple clinical setting (small/moderate sample size and small number of predictors) with Monte-Carlo simulations. METHODS: Synthetic data (250 or 1000 patients) were generated that closely resembled 5 prognostic factors preselected based on a European Osteosarcoma Intergroup study (MRC BO06/EORTC 80931). Comparison was performed between 2 partial logistic artificial neural networks (PLANNs) and Cox models for 20, 40, 61, and 80% censoring. Survival times were generated from a log-normal distribution. Models were contrasted in terms of the C-index, Brier score at 0-5 years, integrated Brier score (IBS) at 5 years, and miscalibration at 2 and 5 years (usually neglected). The endpoint of interest was overall survival. RESULTS: PLANNs original/extended were tuned based on the IBS at 5 years and the C-index, achieving a slightly better performance with the IBS. Comparison with Cox models showed that PLANNs can reach similar predictive performance on simulated data for most scenarios with respect to the C-index, Brier score, or IBS. However, Cox models were frequently less miscalibrated. Performance was robust in scenario data where censored patients were removed before 2 years or curtailing at 5 years was performed (on training data). CONCLUSION: Survival neural networks reached a comparable predictive performance with Cox models but were generally less well calibrated. All in all, researchers should be aware of burdensome aspects of ML techniques such as data preprocessing, tuning of hyperparameters, and computational intensity that render them disadvantageous against conventional regression models in a simple clinical setting.

From Oncological Paradigms to Non-Communicable Disease Pandemic. The Need of Recovery Human Biology Evolution.

The paradigm of the Somatic Mutation Theory (SMT) is failing, and a new paradigm is underway but not yet established. What is being challenged is a conceptual approach that involves the entire human biology and the development of chronic diseases. The behavior of breast and other solid cancers is compatible with the concept that the primary tumor is able to control its microscopic metastases, in the same way that an organ (e.g., the liver) is able to control its physiological size. This finding suggested that cancer and its metastases may behave as an organoid. The new paradigm under construction considers the origin of tumors as a disturbance in the communication network between tissue cell populations and between cells and extracellular matrix, and supports a systemic approach to the study of both healthy and pathologic tissues. The commentary provides a rationale for the role of physical exercise in the control of tumor dormancy according to a human evolutionary perspective.

Late Recurrence in Breast Cancer: To Run after the Oxen or to Try to Close the Barn?

The problem of late recurrence in breast cancer has recently gained attention and was also addressed in an international workshop held in Toronto (ON, Canada), in which several aspects of the question were examined. This Commentary offers a few considerations, which may be useful for the ongoing investigations. A few premises are discussed: (a) clinical recurrences, especially the late ones, imply periods of tumor dormancy; (b) a structured pattern of distant metastases appearance is detectable in both early and late follow-up times; (c) the current general paradigm underlying neoplastic treatments, i.e., that killing all cancer cells is the only way to control the disease, which is strictly sprouting from the somatic mutation theory, should be re-considered. Finally, a few research approaches are suggested.