Complexity in the tumour microenvironment: Cancer associated fibroblast gene expression patterns identify both common and unique features of tumour-stroma crosstalk across cancer types.

Cancer is a complex disease, driven by the accumulation of several somatic aberrations but fostered by a two-way interaction between tumour cells and the surrounding microenvironment. Cancer associated fibroblasts (CAFs) represent one of the major players in tumour-stroma crosstalk. Recent in vitro and in vivo studies, often conducted by employing high throughput approaches, have started unravelling the key pathways involved in their functional effects. This review focus on open challenges in the study of CAF properties and function, highlighting at the same time the existence of common mechanisms as well as peculiarities in different cancer types (breast, prostate and lung cancer). Although still limited by current experimental models, which are unable to deal with the full level of complexity of the tumour microenvironment, a better understanding of these mechanisms may enable the identification of new biomarkers and therapeutic targets, to improve current strategies for cancer diagnosis and treatment.

Gene expression profiling of circulating tumor cells in breast cancer.

BACKGROUND: Determining the transcriptional profile of circulating tumor cells (CTCs) may allow the acquisition of clinically relevant information while overcoming tumor heterogeneity-related biases associated with use of tissue samples for biomarker assessment. However, such molecular characterization is challenging because CTCs are rare and outnumbered by blood cells. METHODS: Here, we describe a technical protocol to measure the expression of >29 000 genes in CTCs captured from whole blood with magnetic beads linked with antibodies against epithelial cell adhesion molecule (EpCAM) and the carcinoma-associated mucin, MUC1, designed to be used for CTC characterization in clinical samples. Low numbers of cells (5-200) from the MCF7 and MDA-MB-468 breast cancer cell lines were spiked in healthy donor blood samples and isolated with the AdnaTest EMT-1/Stem CellSelect kit. Gene expression profiles (GEPs) were obtained with the WG-DASL HT assay and compared with GEPs obtained from RNA isolated from cultured cell lines and unspiked samples. RESULTS: GEPs from samples containing 25 or more spiked cells correlated (r = 0.95) with cognate 100-ng RNA input samples, clustered separately from blood control samples, and allowed MCF7 and MDA-MB-468 cells to be distinguished. GEPs with comparable technical quality were also obtained in a preliminary series of clinical samples. CONCLUSIONS: Our approach allows technically reliable GEPs to be obtained from isolated CTCs for the acquisition of biologically useful information. It is reproducible and suitable for application in prospective studies to assess the clinical utility of CTC GEPs, provided that >25 CTCs can be isolated.

Impact of the First Year of the COVID-19 Pandemic on Pediatric Emergency Department Attendance in a Tertiary Center in South Italy: An Interrupted Time-Series Analysis.

BACKGROUND: The evidence shows a reduction in pediatric emergency department (PED) flows during the early stages of the COVID-19 pandemic. Using interrupted time-series analysis, we evaluated the impact of different stages of the pandemic response on overall and cause-specific PED attendance at a tertiary hospital in south Italy. Our methods included evaluations of total visits, hospitalizations, accesses for critical illnesses and four etiological categories (transmissible and non-transmissible infectious diseases, trauma and mental-health) during March-December 2020, which were compared with analogous intervals from 2016 to 2019; the pandemic period was divided into three segments: the "first lockdown" (FL, 9 March-3 May), the "post-lockdown" (PL, 4 May-6 November) and the "second lockdown" (SL, 7 November-31 December). Our results showed that attendance dropped by a mean of 50.09% during the pandemic stages, while hospitalizations increased. Critical illnesses decreased during FL (incidence rate ratio -IRR- 0.37, 95% CI 0.13, 0.88) e SL (IRR 0.09, 95% CI 0.01, 0.74) and transmissible disease related visits reduced more markedly and persistently (FL: IRR 0.18, 95% CI 0.14, 0.24; PL: IRR 0.20, 95% CI 0.13, 0.31, SL: IRR 0.17, 95% CI 0.10, 0.29). Non-infectious diseases returned to pre-COVID-19 pandemic levels by PL. We concluded that that the results highlight the specific effect of the late 2020 containment measures on transmissible infectious diseases and their burden on pediatric emergency resources. This evidence can inform resource allocation and interventions to mitigate the impact of infectious diseases on pediatric populations and the health-care system.

Evolution of Pediatric Migraine Patients Admitted at an Emergency Department after a 10-Year Follow-Up.

BACKGROUND: Despite its high prevalence, the clinical course of pediatric migraine has not been fully understood, and previous studies present conflicting results. We present here the findings of a 10-year follow-up study involving children with severe migraine pain admitted to our emergency department. Furthermore, all studies were carried out on selected outpatient clinical case studies. Our aim was to evaluate a population of migraine children admitted to an emergency department because of increased severity or frequency of pain or even because of very anxious parents concerning their child's headache in order to describe their long-term outcomes, whether it differed from that of outpatient populations and to identify possible predictors of prognosis. METHODS: We recruited 80 subjects with migraine headaches (mean age 8 years with a range of 4-14 years, 50% females), attending the baseline examination of a population admitted for a headache to the Emergency Department in the first half year of 2012. Of the 80 subjects, 48 (60%) were eligible for follow-up in 2022. We included in our study only patients diagnosed with migraine, according to the diagnostic criteria of the International Classification of Headache Disorders. All were contacted by telephone, and a semi-structured questionnaire was provided to them by email. The association between several possible prognostic factors (gender, familiar neurologic disorders, prenatal and perinatal disorders, social activities, sleep disorders, etc.) and the long-term persistence of migraine headaches were explored using logistic regression analysis. RESULTS: Of 48 subjects with migraine headaches at baseline, 31 (65%) had persistent migraine, and 17 (35%) experienced remission. The preliminary results showed that the presence of neurologic disorders in parents (p < 0.01-odds ratio 9.34 (2.53-41.64) and sleep disorders (p < 0.01-odds ratio 13.18 (2.25-252.74) significantly predicted the 10-year persistence of migraine headaches, while the other considered predictors were found not to influence prognosis. CONCLUSIONS: To our knowledge, this was the first study conducted on a selected pediatric population upon admission to the emergency room. Our study suggests that a population of pediatric migraine selected for admission to the emergency department also shows a favorable long-term prognosis, like the studies conducted in the outpatient sample. Familial neurological comorbidity and sleep disorders were unfavorable factors for predicting good outcomes.

Prognostic and functional role of subtype-specific tumor-stroma interaction in breast cancer.

None of the clinically relevant gene expression signatures available for breast cancer were specifically developed to capture the influence of the microenvironment on tumor cells. Here, we attempted to build subtype-specific signatures derived from an in vitro model reproducing tumor cell modifications after interaction with activated or normal stromal cells. Gene expression signatures derived from HER2+, luminal, and basal breast cancer cell lines (treated by normal fibroblasts or cancer-associated fibroblasts conditioned media) were evaluated in clinical tumors by in silico analysis on published gene expression profiles (GEPs). Patients were classified as microenvironment-positive (µENV+ve), that is, with tumors showing molecular profiles suggesting activation by the stroma, or microenvironment-negative (µENV-ve) based on correlation of their tumors' GEP with the respective subtype-specific signature. Patients with estrogen receptor alpha (ER)+/HER2-/µENV+ve tumors were characterized by 2.5-fold higher risk of developing distant metastases (HR = 2.546; 95% CI: 1.751-3.701, P = 9.84E-07), while µENV status did not affect, or only suggested the risk of distant metastases, in women with HER2+ (HR = 1.541; 95% CI: 0.788-3.012, P = 0.206) or ER-/HER2- tumors (HR = 1.894; 95% CI: 0.938-3.824; P = 0.0747), respectively. In ER+/HER2- tumors, the µENV status remained significantly associated with metastatic progression (HR = 2.098; CI: 1.214-3.624; P = 0.00791) in multivariable analysis including size, age, and Genomic Grade Index. Validity of our in vitro model was also supported by in vitro biological endpoints such as cell growth (MTT assay) and migration/invasion (Transwell assay). In vitro-derived gene signatures tracing the bidirectional interaction with cancer activated fibroblasts are subtype-specific and add independent prognostic information to classical prognostic variables in women with ER+/HER2- tumors.

Subtype-Specific Metagene-Based Prediction of Outcome after Neoadjuvant and Adjuvant Treatment in Breast Cancer.

PURPOSE: In spite of improvements of average benefit from adjuvant/neoadjuvant treatments, there are still individual patients with early breast cancer at high risk of relapse. We explored the association with outcome of robust gene cluster-based metagenes linked to proliferation, ER-related genes, and immune response to identify those high-risk patients. EXPERIMENTAL DESIGN: A total of 3,847 publicly available gene-expression profiles were analyzed (untreated, N = 826; tamoxifen-treated, N = 685; chemotherapy-treated, N = 1,150). Genes poorly performing in formalin-fixed samples were removed. Outcomes of interest were pathologic-complete response (pCR) and distant metastasis-free survival (DMFS). In ER(+)HER2(-), the proliferation and ER-related metagenes were combined to define three risk groups. In HER2(+) and ER(-)HER2(-) risk groups were defined by tertiles of an immune-related metagene. RESULTS: The high-proliferation/low-ER group of ER(+)HER2(-) breast cancer had significantly higher pCR rate [OR, 5.01 (1.76-17.99), P = 0.005], but poorer outcome [HR = 3.73 (1.63-8.51), P = 0.0018] than the low-proliferation/high-ER. A similar association with outcome applied to patients with residual disease (RD) after neoadjuvant chemotherapy (P = 0.01). In ER(-)HER2(-) and HER2(+) breast cancer, immune metagene in the high tertile was linked to higher pCR [33.7% vs. 11.6% in high and low tertile, respectively; OR, 3.87 (1.79-8.95); P = 0.0009]. In ER(-)HER2(-), after adjuvant/neoadjuvant chemotherapy, 5-year DMFS was 85.4% for high-tertile immune metagene, and 43.9% for low tertile. The outcome association was similar in patients with RD (P = 0.0055). In HER2(+) breast cancer treated with chemotherapy the association with risk of relapse was not significant. CONCLUSIONS: We developed metagene-based predictors able to define low and high risk of relapse after adjuvant/neoadjuvant therapy. High-risk patients so defined should be preferably considered for trials with investigational agents.

Integrated gene and miRNA expression analysis of prostate cancer associated fibroblasts supports a prominent role for interleukin-6 in fibroblast activation.

Tumor microenvironment coevolves with and simultaneously sustains cancer progression. In prostate carcinoma (PCa), cancer associated fibroblasts (CAF) have been shown to fuel tumor development and metastasis by mutually interacting with tumor cells. Molecular mechanisms leading to activation of CAFs from tissue-resident fibroblasts, circulating bone marrow-derived fibroblast progenitors or mesenchymal stem cells are largely unknown. Through integrated gene and microRNA expression profiling, we showed that PCa-derived CAF transcriptome strictly resembles that of normal fibroblasts stimulated in vitro with interleukin-6 (IL6), thus proving evidence, for the first time, that the cytokine is able per se to induce most of the transcriptional changes characteristic of patient-derived CAFs. Comparison with publicly available datasets, however, suggested that prostate CAFs may be alternatively characterized by IL6 and TGFß-related signatures, indicating that either signal, depending on the context, may concur to fibroblast activation. Our analyses also highlighted novel pathways potentially relevant for induction of a reactive stroma. In addition, we revealed a role for muscle-specific miR-133b as a soluble factor secreted by activated fibroblasts to support paracrine activation of non-activated fibroblasts or promote tumor progression.Overall, we provided insights into the molecular mechanisms driving fibroblast activation in PCa, thus contributing to identify novel hits for the development of therapeutic strategies targeting the crucial interplay between tumor cells and their microenvironment.