microRNAs combined to radiomic features as a predictor of complete clinical response after neoadjuvant radio-chemotherapy for locally advanced rectal cancer: a preliminary study.

OBJECTIVE: To define a predictive Artificial Intelligence (AI) algorithm based on the integration of a set of biopsy-based microRNAs expression data and radiomic features to understand their potential impact in predicting clinical response (CR) to neoadjuvant radio-chemotherapy (nRCT). The identification of patients who would truly benefit from nRCT for Locally Advanced Rectal Cancer (LARC) could be crucial for an improvement in a tailored therapy. METHODS: Forty patients with LARC were retrospectively analyzed. An MRI of the pelvis before and after nRCT was performed. In the diagnostic biopsy, the expression levels of 7 miRNAs were measured and correlated with the tumor response rate (TRG), assessed on the surgical sample. The accuracy of complete CR (cCR) prediction was compared for i) clinical predictors; ii) radiomic features; iii) miRNAs levels; and iv) combination of radiomics and miRNAs. RESULTS: Clinical predictors showed the lowest accuracy. The best performing model was based on the integration of radiomic features with miR-145 expression level (AUC-ROC = 0.90). AI algorithm, based on radiomics features and the overexpression of miR-145, showed an association with the TRG class and demonstrated a significant impact on the outcome. CONCLUSION: The pre-treatment identification of responders/NON-responders to nRCT could address patients to a personalized strategy, such as total neoadjuvant therapy (TNT) for responders and upfront surgery for non-responders. The combination of radiomic features and miRNAs expression data from images and biopsy obtained through standard of care has the potential to accelerate the discovery of a noninvasive multimodal approach to predict the cCR after nRCT for LARC.

Cyclin E1 and Rb modulation as common events at time of resistance to palbociclib in hormone receptor-positive breast cancer.

CDK4/6 inhibitors represent a new treatment standard for hormone receptor-positive (HR+), HER2-negative advanced breast cancer (BC) patients. Although efficacious, resistance to these agents is universal. Here, we profiled a large panel of HR+ BC cell lines with conditioned resistance to the CDK4/6 inhibitor palbociclib, and analyzed cell cycle-related markers by gene expression profiles (GEP) and western blot (WB). GEP showed high molecular heterogeneity among the models, with E2F targets being significantly enriched both during treatment and at the time of resistance. By both WB and GEP, a common molecular feature at the time of palbociclib resistance was the concomitant overexpression of cyclin E1 and down-regulation of Rb. CCNE1 was the only significantly up-regulated gene among E2F targets at resistance with CCNE1 genomic amplification being observed in two resistant models. Rb was downregulated in all resistant models; a reduction of RB1 copy number was observed in three resistant cell lines. In silico analyses showed that CCNE1/RB1 ratio correlated with palbociclib IC50 in different datasets of both breast and non-breast cancer cell lines, performing better than CCNE1 or RB1 taken separately. Finally, the CCNE1/RB1 ratio was shown to be an adverse prognostic factor in patients with ER+ BC and to be able to discriminate palbociclib-sensitive versus resistant among patients enrolled in the NeoPalAna trial, a neoadjuvant trial testing palbociclib, performing better than CCNE1 or RB1 alone. Our data suggest that the CCNE1/RB1 ratio may be a viable biomarker of palbociclib resistance, warranting further clinical validation.

A gene expression signature of retinoblastoma loss-of-function is a predictive biomarker of resistance to palbociclib in breast cancer cell lines and is prognostic in patients with ER positive early breast cancer.

Palbociclib is a CDK4/6 inhibitor that received FDA approval for treatment of hormone receptor positive (HR+) HER2 negative (HER2neg) advanced breast cancer. To better personalize patients treatment it is critical to identify subgroups that would mostly benefit from it. We hypothesize that complex alterations of the Retinoblastoma (Rb) pathway might be implicated in resistance to CDK4/6 inhibitors and aim to investigate whether signatures of Rb loss-of-function would identify breast cancer cell lines resistant to palbociclib. We established a gene expression signature of Rb loss-of-function (RBsig) by identifying genes correlated with E2F1 and E2F2 expression in breast cancers within The Cancer Genome Atlas. We assessed the RBsig prognostic role in the METABRIC and in a comprehensive breast cancer meta-dataset. Finally, we analyzed whether RBsig would discriminate palbociclib-sensitive and -resistant breast cancer cells in a large RNA sequencing-based dataset. The RBsig was associated with RB1 genetic status in all tumors (p <7e-32) and in luminal or basal subtypes (p < 7e-11 and p < 0.002, respectively). The RBsig was prognostic in the METABRIC dataset (discovery: HR = 1.93 [1.5-2.4] p = 1.4e-08; validation: HR = 2.01 [1.6-2.5] p = 1.3e-09). Untreated and endocrine treated patients with estrogen receptor positive breast cancer expressing high RBsig had significantly worse recurrence free survival compared to those with low RBsig (HR = 2.37 [1.8 - 3.2] p = 1.87e-08 and HR = 2.62 [1.9- 3.5] p = 8.6e-11, respectively). The RBsig was able to identify palbociclib resistant and sensitive breast cancer cells (ROC AUC = 0,7778). Signatures of RB loss might be helpful in personalizing treatment of patients with HR+/HER2neg breast cancer. Further validation in patients receiving palbociclib is warranted.

Specific mesothelial signature marks the heterogeneity of mesenchymal stem cells from high-grade serous ovarian cancer.

Mesenchymal stem/stromal cells (MSCs) are the precursors of various cell types that compose both normal and cancer tissue microenvironments. In order to support the widely diversified parenchymal cells and tissue organization, MSCs are characterized by a large degree of heterogeneity, although available analyses of molecular and transcriptional data do not provide clear evidence. We have isolated MSCs from high-grade serous ovarian cancers (HG-SOCs) and various normal tissues (N-MSCs), demonstrated their normal genotype and analyzed their transcriptional activity with respect to the large comprehensive FANTOM5 sample dataset. Our integrative analysis conducted against the extensive panel of primary cells and tissues of the FANTOM5 project allowed us to mark the HG-SOC-MSCs CAGE-seq transcriptional heterogeneity and to identify a cell-type-specific transcriptional activity showing a significant relationship with primary mesothelial cells. Our analysis shows that MSCs isolated from different tissues are highly heterogeneous. The mesothelial-related gene signature identified in this study supports the hypothesis that HG-SOC-MSCs are bona fide representatives of the ovarian district. This finding indicates that HG-SOC-MSCs could actually derive from the coelomic mesothelium, suggesting that they might be linked to the epithelial tumor through common embryological precursors.

Effects of age and heart failure on human cardiac stem cell function.

Currently, it is unknown whether defects in stem cell growth and differentiation contribute to myocardial aging and chronic heart failure (CHF), and whether a compartment of functional human cardiac stem cells (hCSCs) persists in the decompensated heart. To determine whether aging and CHF are critical determinants of the loss in growth reserve of the heart, the properties of hCSCs were evaluated in 18 control and 23 explanted hearts. Age and CHF showed a progressive decrease in functionally competent hCSCs. Chronological age was a major predictor of five biomarkers of hCSC senescence: telomeric shortening, attenuated telomerase activity, telomere dysfunction-induced foci, and p21(Cip1) and p16(INK4a) expression. CHF had similar consequences for hCSCs, suggesting that defects in the balance between cardiomyocyte mass and the pool of nonsenescent hCSCs may condition the evolution of the decompensated myopathy. A correlation was found previously between telomere length in circulating bone marrow cells and cardiovascular diseases, but that analysis was restricted to average telomere length in a cell population, neglecting the fact that telomere attrition does not occur uniformly in all cells. The present study provides the first demonstration that dysfunctional telomeres in hCSCs are biomarkers of aging and heart failure. The biomarkers of cellular senescence identified here can be used to define the birth date of hCSCs and to sort young cells with potential therapeutic efficacy.

Multipotent progenitor cells are present in human peripheral blood.

To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of approximately 3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow).

The aims of our study were to verify whether it was possible to generate in vitro, from different adult human tissues, a population of cells that behaved, in culture, as multipotent stem cells and if these latter shared common properties. To this purpose, we grew and cloned finite cell lines obtained from adult human liver, heart, and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs, obtained from the 3 different tissues, expressed the pluripotent state-specific transcription factors Oct-4, NANOG, and REX1, displayed telomerase activity, and exhibited a wide range of differentiation potential, as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content, and shared a common gene expression signature, compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular, the pathways regulating stem cell self-renewal/maintenance, such as Wnt, Hedgehog, and Notch, were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and functional features of mature cells even embryologically not related to the tissue of origin.