Economic preferences in the health setting. Three case studies concerning the needs of breast cancer patients, pain management in oncology and extreme end-of-life decisions

The thesis describes three studies concerning the role of the Economic Preference set investigated in the Global Preference Survey (GPS) in the following cases: 1) the needs of women with breast cancer; 2) pain undertreament in oncology; 3) legal status of euthanasia and assisted suicide. The analyses, based on regression techniques, were always conducted on the basis of aggregate data and revealed in all cases a possible role of the Economic Preferences studied, also resisting the concomitant effect of the other covariates that were considered from time to time. Regarding individual studies, the related conclusion are: 1) Economic Preferences appear to play a role in influencing the needs of women with breast cancer, albeit of non-trivial interpretation, statistically "resisting" the concomitant effect of the other independent variables considered. However, these results should be considered preliminary and need further confirmation, possibly with prospective studies conducted at the level of the individual; 2) the results show a good degree of internal consistency with regard to pro-social GPS scores, since they are all found to be non-statistically significant and united, albeit only weakly in trend, by a negative correlation with the % of pain undertreated patients. Sharper, at least statistically, is the role of Patience and Willingness to Take Risk, although of more complex empirical interpretation. 3) the results seem to indicate an obvious role of Economic Preferences, however difficult to interpret empirically. Less evidence, at least on the inferential level, emerged, however, regarding variables that, based on common sense, should play an even more obvious role than Economic Preferences in orienting attitudes toward euthanasia and assisted suicide, namely Healthcare System, Legal Origin, and Kinship Tightness; striking, in particular, is the inability to prove a role for the dominant religious orientation even with a simple bivariate analysis.

Clinical impact of next-generation sequencing multi-gene panel highlighting the landscape of germline alterations in ovarian cancer patients

BRCA1 and BRCA2 are the most frequently mutated genes in ovarian cancer (OC), crucial both for the identification of cancer predisposition and therapeutic choices. However, germline variants in other genes could be involved in OC susceptibility. We characterized OC patients to detect mutations in genes other than BRCA1/2 that could be associated with a high risk to develop OC, and that could permit patients to enter the most appropriate treatment and surveillance program. Next-Generation Sequencing analysis with a 94-gene panel was performed on germline DNA of 219 OC patients. We identified 34 pathogenic/likely-pathogenic variants in BRCA1/2 and 38 in other 21 genes. Patients with pathogenic/likely-pathogenic variants in non-BRCA1/2 genes developed mainly OC alone compared to the other groups that developed also breast cancer or other tumors (p=0.001). Clinical correlation analysis showed that low-risk patients were significantly associated with platinum sensitivity (p<0.001). Regarding PARP inhibitors (PARPi) response, patients with pathogenic mutations in non-BRCA1/2 genes had significantly worse PFS and OS. Moreover, a statistically significant worse PFS was found for every increase of one thousand platelets before PARPi treatment. To conclude, knowledge about molecular alterations in genes beyond BRCA1/2 in OC could allow for more personalized diagnostic, predictive, prognostic, and therapeutic strategies for OC patients.

Phenotypic and molecular investigation of circulating tumor cells (CTCs) isolated from breast cancer patients at single cell resolution

Despite the paramount advances in cancer research, breast cancer (BC) still ranks one of the leading causes of cancer-related death worldwide. Thanks to the screening campaign started in developed countries, BC is often diagnosed at early stages (non-metastatic BC, nmBC), but disease relapse occurrence even after decades and at distant sites is not an uncommon phenomenon. Conversely, metastatic BC (mBC) is considered an incurable disease. The major perpetrators of tumor spread to secondary organs are circulating tumor cells (CTCs), a rare population of cells detectable in the peripheral blood of oncologic patients. In this study, CTCs from patients diagnosed with luminal nmBC and mBC (hormone receptor positive, Human Epidermal Growth Factor Receptor 2 (HER2) negative) were characterized at both phenotypic and molecular levels. To better understand the molecular mechanisms underlying their biology and their metastatic potential, next-generation sequencing (NGS) analyses were performed at single-cell resolution to assess copy number aberrations (CNAs), single nucleotide variants (SNVs) and gene expression profiling. The findings of this study arise hints in CTC detection, and pave the way to new application in CTC research.

Evaluation of Antitumoral activity of bone targeted drugs/conventional chemotherapies and identification of biomarkers for the selection of patients with breast cancer for the bone targeted therapy in adjuvant setting

Bone metastases are responsible for different clinical complications defined as skeletal-related events (SREs) such as pathologic fractures, spinal cord compression, hypercalcaemia, bone marrow infiltration and severe bone pain requiring palliative radiotherapy. The general aim of these three years research period was to improve the management of patients with bone metastases through two different approaches of translational research. Firstly in vitro preclinical tests were conducted on breast cancer cells and on indirect co-colture of cancer cells and osteoclasts to evaluate bone targeted therapy singly and in combination with conventional chemotherapy. The study suggests that zoledronic acid has an antitumor activity in breast cancer cell lines. Its mechanism of action involves the decrease of RAS and RHO, as in osteoclasts. Repeated treatment enhances antitumor activity compared to non-repeated treatment. Furthermore the combination Zoledronic Acid + Cisplatin induced a high antitumoral activity in the two triple-negative lines MDA-MB-231 and BRC-230. The p21, pMAPK and m-TOR pathways were regulated by this combined treatment, particularly at lower Cisplatin doses. A co-colture system to test the activity of bone-targeted molecules on monocytes-breast conditioned by breast cancer cells was also developed. Another important criticism of the treatment of breast cancer patients, is the selection of patients who will benefit of bone targeted therapy in the adjuvant setting. A retrospective case-control study on breast cancer patients to find new predictive markers of bone metastases in the primary tumors was performed. Eight markers were evaluated and TFF1 and CXCR4 were found to discriminate between patients with relapse to bone respect to patients with no evidence of disease. In particular TFF1 was the most accurate marker reaching a sensitivity of 63% and a specificity of 79%. This marker could be a useful tool for clinicians to select patients who could benefit for bone targeted therapy in adjuvant setting.

The role of interleuchin 6 (IL-6) in the promotion of an aggressive and stem cell-like phenotype in human breast cancer cells and in stem/progenitor cells expanded in vitro as mammospheres

High serum levels of Interleukin-6 (IL-6) correlate with poor outcome in breast cancer patients. However no data are available on the relationship between IL-6 and stem/progenitor cells which may fuel the genesis of breast cancer in vivo. Herein, we address this issue in mammospheres (MS), multi-cellular structures enriched in stem/progenitor cells of the mammary gland, and also in MCF-7 breast cancer cells. We show that MS from node invasive breast carcinoma tissues express IL-6 mRNA at higher levels than MS from matched non-neoplastic mammary glands. We find that IL-6 mRNA is detectable only in basal-like breast carcinoma tissues, an aggressive variant showing stem cell features. Our results reveal that IL-6 triggers a Notch-3-dependent up-regulation of the Notch ligand Jagged-1, whose interaction with Notch-3 promotes the growth of MS and MCF-7 derived spheroids. Moreover, IL-6 induces a Notch-3-dependent up-regulation of the carbonic anhydrase IX gene, which promotes a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. Finally, an autocrine IL-6 loop relies upon Notch-3 activity to sustain the aggressive features of MCF-7-derived hypoxia-selected cells. In conclusion, our data support the hypothesis that IL-6 induces malignant features in Notch-3 expressing, stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

Role of new molecular approaches for the early diagnosis of bladder cancer in clinical practice

There is an urgent need to improve the performance of urine cytology for the diagnosis of bladder cancer. In preliminary studies, telomerase activity evaluated by telomeric repeat amplification protocol (TRAP) assay and chromosomal aneuploidy detected by fluorescence in situ hybridization (FISH) in the diagnosis of bladder cancer have produced important results. Urine cell-free (UCF) DNA has also been proposed as a potential marker for early bladder cancer diagnosis. In the first study the diagnostic performance of TRAP assay and FISH analysis was assessed, while the second study evaluated the potential role of UCF DNA integrity in early bladder cancer diagnosis. In the first cross-sectional study, 289 consecutive patients who presented with urinary symptoms underwent cystoscopy and cytology evaluation. In the second study, UCF DNA was isolated from 51 bladder cancer patients, 46 symptomatic patients, and 32 healthy volunteers. c-Myc, BCAS1 and HER2 gene sequences longer than 250 bp were quantified by real time PCR to verify UCF DNA integrity. In the first study, sensitivity and specificity were 0.39 and 0.83, respectively, for cytology; 0.66 and 0.72 for TRAP; 0.78 and 0.60 for the cytology and TRAP combination; 0.78 and 0.78 for the cytology, TRAP and FISH combination; and 0.65 and 0.93 for the TRAP and FISH combination. In the second study, at the best cutoff of 0.1 ng/µl, UCF DNA integrity analysis showed a sensitivity of 0.73 and a specificity of 0.84 in healthy individuals and 0.83 in symptomatic patients. The preliminary results suggest that these biomarkers could potentially be used for the early diagnosis of bladder cancer, especially in high-risk populations (e.g, symptomatic individuals exposed to occupational risk) who may benefit from the use of noninvasive diagnostic tests in terms of cost-benefit.

Preclinical development of anti-cancer strategies against human HER-2

HER-2 is a 185 kDa transmembrane receptor tyrosine kinase that belongs to the EGFR family. HER-2 is overexpressed in nearly 25% of human breast cancers and women with this subtype of breast cancer have a worse prognosis and frequently develop metastases. The progressive high number of HER-2-positive breast cancer patients with metastatic spread in the brain (up to half of women) has been attributed to the reduction in mortality, the effectiveness of Trastuzumab in killing metastatic cells in other organs and to its incapability to cross the blood-brain barrier. Apart from full-length-HER-2, a splice variant of HER-2 lacking exon 16 (here referred to as D16) was identified in human HER-2-positive breast cancers. Here, the contribution of HER-2 and D16 to mammary carcinogenesis was investigated in a model transgenic for both genes (F1 model). A dominant role of D16, especially in early stages of tumorigenesis, was suggested and the coexistence of heterogeneous levels of HER-2 and D16 in F1 tumors revealed the undeniable value of F1 strain as preclinical model of HER-2-positive breast cancer, closer resembling the human situation in respect to previous models. The therapeutical efficacy of anti-HER-2 agents, targeting HER-2 receptor (Trastuzumab, Lapatinib, R-LM249) or signaling effectors (Dasatinib, UO126, NVP-BKM120), was investigated in models of local or advanced HER-2-positive breast cancer. In contrast with early studies, data herein collected suggested that the presence of D16 can predict a better response to Trastuzumab and other agents targeting HER-2 receptor or Src activity. Using a multiorgan HER-2-positive metastatic model, the efficacy of NVP-BKM120 (PI3K inhibitor) in blocking the growth of brain metastases and the oncolytic ability of R-LM249 (HER-2-retargeted HSV) to reach and destroy metastatic HER-2-positive cancer cells were shown. Finally, exploiting the definition of “oncoantigen” given to HER-2, the immunopreventive activity of two vaccines on HER-2-positive mammary tumorigenesis was demonstrated.

Unbalanced R-loops and micronuclei induced by DNA topoisomerase I poisons in cancer cells

Topoisomerase I (Top1) poisons are among the most clinically-effective drugs used for colon, ovary and lung cancers. Unpublished data from our lab have recently revealed that the structurally-unrelated Top1 poisons, Camptothecin (CPT) and Indimitecan (LMP776), induce the formation of micronuclei (MNi) in human cancer cells. In addition, MNi trigger an innate immune gene response by stimulating the cGAS/STING pathway. As the mechanisms of MNi formation are not fully determined, our aim is here to establish how MNi form after Top1 poisoning. Using immunofluorescence assays and EdU labelling of nascent DNAs, our results show that, after 24 hours of recovery, a short treatment with sub-cytotoxic doses of Top1 poisons induces the formation of MNi that do not contain newly synthetized (EdU+) DNA. We also saw that Top1 poisons delay replication machinery reducing EdU incorporation and produce significant levels of the damage markers γH2AX and p53BP1 in S-phase cells but not in G1 and G2/M cells. The results also show that MNi formation is dependent on R-loops, as RNaseH1 overexpression markedly reduces Top1 induced MNi. Genome-wide mapping of R-loops by DRIP-seq technique revealed that R-loop levels are both decreased and increased by CPT. In particular, increased R-loops are mainly found at active genes and always overlapped with Top1cc sites. We also found that increased R-loops overlap with lamina-associated chromatin domains while decreased R-loops correlate with replication origin sites. Overall, our data are consistent with the formation of MNi due to R-loop increase and under-replication at specific regions caused by Top1 poisons. These results will eventually help in developing new strategies for effective personalized interventions by using Top1-targeted compounds as immuno-modulators in cancer patients.

Ultra-hypofractionated stereotactic radiotherapy for prostate cancer in 5 fractions: current evidences and our clinical experience

AIMS: The present is a retrospective evaluation of acute genito-urinary (GU) and gastro-intestinal (GI) toxicity, in addition to biochemical recurrence rate in 57 prostate cancer patients treated at our Institution with ultra-hypofractionated RT (UHRT) schedule. METHODS: From January 2021 to December 2022 we have treated 57 patients with prostate cancer, using an UHRT scheme of 5-fractions every other day for a total dose delivered of 36.25 Gy, according to the PACE-B trial treatment schedule. Good urinary function, assessed by International Prostate Symptom Score (IPSS), were required. The simulation CT scans were acquired in supine position and fused with MRI for CTVs definition for every patient. Each treatment was performed by Accuray's TomoTherapy with daily IGRT. The evaluation of the set-up was very restrictive before daily treatment delivery. RESULTS: According to RTOG toxicity scale, the acute GU toxicity at 3 months from RT, GU toxicity was G0 for 30 patients (52.6%), G1 for 26 (45.6%) and G2 for one only (1.75%); rectal toxicity was G0 for 56 patients (98.25%) and G1 for one only (1.75%). The median follow-up (FU) was 9 months (2-24 months). In the following FU months, we observed progressively lower urinary and rectal toxicity, except for one patient who showed G2 GU toxicity at 12 months. All but one patient had a progressive PSA value decrease. CONCLUSIONS: In our experience, UHRT appears to be safe and well tolerated even without the use of rectal spacer devices. A longer FU is necessary to evaluate late toxicity and disease control rate.