The inhibition of aerobic glycolysis as a therapeutic approach to improve cancer chemotherapy

The aim of the research project discussed in this thesis was to study the inhibition of aerobic glycolysis, that is the metabolic pathway exploited by cancer cells for the ATP generation. This observation has led to the evaluation of glycolytic inhibitors as potential anticancer agents. Lactate dehydrogenase (LDH) is the only enzyme whose inhibition should allow a blocking of aerobic glycolysis of tumor cells without damaging the normal cells which, in conditions of normal functional activity and sufficient oxygen supply, do not need this enzyme. In preliminar experiments we demonstrated that oxamic acid and tartronic acid, two LDH competitive inhibitors, impaired aerobic glycolysis and replication of cells from human hepatocellular carcinoma. Therefore, we proposed that the depletion of ATP levels in neoplastic cells, could improved the chemotherapeutic index of associated anticancer drugs; in particular, it was studied the association of oxamic acid and multi-targeted kinase inhibitors. A synergistic effect in combination with sorafenib was observed, and we demonstrated that this was related to the capacity of sorafenib to hinder the oxidative phosphorylation, so that cells were more dependent to aerobic glycolysis. These results linked to LDH blockage encouraged us to search for LDH inhibitors more powerful than oxamic acid; thus, in collaboration with the Department of Pharmaceutical Sciences of Bologna University we identified a new molecule, galloflavin, able to inhibit both A and B isoforms of LDH enzyme. The effects of galloflavin were studied on different human cancer cell lines (hepatocellular carcinoma, breast cancer, Burkitt’s lymphoma). Although exhibiting different power on the tested cell lines, galloflavin was constantly found to inhibit lactate and ATP production and to induce cell death, mainly in the form of apoptosis. Finally, as LDH-A is able to bind single stranded DNA, thus stimulating cell transcription, galloflavin effects were also studied on this other LDH function.

RNA Interference and cyclooxygenase-2 (COX-2) regulation in colon cancer cells

Despite new methods and combined strategies, conventional cancer chemotherapy still lacks specificity and induces drug resistance. Gene therapy can offer the potential to obtain the success in the clinical treatment of cancer and this can be achieved by replacing mutated tumour suppressor genes, inhibiting gene transcription, introducing new genes encoding for therapeutic products, or specifically silencing any given target gene. Concerning gene silencing, attention has recently shifted onto the RNA interference (RNAi) phenomenon. Gene silencing mediated by RNAi machinery is based on short RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), that are fully o partially homologous to the mRNA of the genes being silenced, respectively. On one hand, synthetic siRNAs appear as an important research tool to understand the function of a gene and the prospect of using siRNAs as potent and specific inhibitors of any target gene provides a new therapeutical approach for many untreatable diseases, particularly cancer. On the other hand, the discovery of the gene regulatory pathways mediated by miRNAs, offered to the research community new important perspectives for the comprehension of the physiological and, above all, the pathological mechanisms underlying the gene regulation. Indeed, changes in miRNAs expression have been identified in several types of neoplasia and it has also been proposed that the overexpression of genes in cancer cells may be due to the disruption of a control network in which relevant miRNA are implicated. For these reasons, I focused my research on a possible link between RNAi and the enzyme cyclooxygenase-2 (COX-2) in the field of colorectal cancer (CRC), since it has been established that the transition adenoma-adenocarcinoma and the progression of CRC depend on aberrant constitutive expression of COX-2 gene. In fact, overexpressed COX-2 is involved in the block of apoptosis, the stimulation of tumor-angiogenesis and promotes cell invasion, tumour growth and metastatization. On the basis of data reported in the literature, the first aim of my research was to develop an innovative and effective tool, based on the RNAi mechanism, able to silence strongly and specifically COX-2 expression in human colorectal cancer cell lines. In this study, I firstly show that an siRNA sequence directed against COX-2 mRNA (siCOX-2), potently downregulated COX-2 gene expression in human umbilical vein endothelial cells (HUVEC) and inhibited PMA-induced angiogenesis in vitro in a specific, non-toxic manner. Moreover, I found that the insertion of a specific cassette carrying anti-COX-2 shRNA sequence (shCOX-2, the precursor of siCOX-2 previously tested) into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT-29) without activating any interferon response. Phenotypically, COX-2 deficient HT-29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, results reported here indicate an easy-to-use, powerful and high selective virus-based method to knockdown COX-2 gene in a stable and long-lasting manner, in colon cancer cells. Furthermore, they open up the possibility of an in vivo application of this anti-COX-2 retroviral vector, as therapeutic agent for human cancers overexpressing COX-2. In order to improve the tumour selectivity, pSUPER.retro vector was modified for the shCOX-2 expression cassette. The aim was to obtain a strong, specific transcription of shCOX-2 followed by COX-2 silencing mediated by siCOX-2 only in cancer cells. For this reason, H1 promoter in basic pSUPER.retro vector [pS(H1)] was substituted with the human Cox-2 promoter [pS(COX2)] and with a promoter containing repeated copies of the TCF binding element (TBE) [pS(TBE)]. These promoters were choosen because they are partculary activated in colon cancer cells. COX-2 was effectively silenced in HT-29 and HCA-7 colon cancer cells by using enhanced pS(COX2) and pS(TBE) vectors. In particular, an higher siCOX-2 production followed by a stronger inhibition of Cox-2 gene were achieved by using pS(TBE) vector, that represents not only the most effective, but also the most specific system to downregulate COX-2 in colon cancer cells. Because of the many limits that a retroviral therapy could have in a possible in vivo treatment of CRC, the next goal was to render the enhanced RNAi-mediate COX-2 silencing more suitable for this kind of application. Xiang and et al. (2006) demonstrated that it is possible to induce RNAi in mammalian cells after infection with engineered E. Coli strains expressing Inv and HlyA genes, which encode for two bacterial factors needed for successful transfer of shRNA in mammalian cells. This system, called “trans-kingdom” RNAi (tkRNAi) could represent an optimal approach for the treatment of colorectal cancer, since E. Coli in normally resident in human intestinal flora and could easily vehicled to the tumor tissue. For this reason, I tested the improved COX-2 silencing mediated by pS(COX2) and pS(TBE) vectors by using tkRNAi system. Results obtained in HT-29 and HCA-7 cell lines were in high agreement with data previously collected after the transfection of pS(COX2) and pS(TBE) vectors in the same cell lines. These findings suggest that tkRNAi system for COX-2 silencing, in particular mediated by pS(TBE) vector, could represent a promising tool for the treatment of colorectal cancer. Flanking the studies addressed to the setting-up of a RNAi-mediated therapeutical strategy, I proposed to get ahead with the comprehension of new molecular basis of human colorectal cancer. In particular, it is known that components of the miRNA/RNAi pathway may be altered during the progressive development of colorectal cancer (CRC), and it has been already demonstrated that some miRNAs work as tumor suppressors or oncomiRs in colon cancer. Thus, my hypothesis was that overexpressed COX-2 protein in colon cancer could be the result of decreased levels of one or more tumor suppressor miRNAs. In this thesis, I clearly show an inverse correlation between COX-2 expression and the human miR- 101(1) levels in colon cancer cell lines, tissues and metastases. I also demonstrate that the in vitro modulating of miR-101(1) expression in colon cancer cell lines leads to significant variations in COX-2 expression, and this phenomenon is based on a direct interaction between miR-101(1) and COX-2 mRNA. Moreover, I started to investigate miR-101(1) regulation in the hypoxic environment since adaptation to hypoxia is critical for tumor cell growth and survival and it is known that COX-2 can be induced directly by hypoxia-inducible factor 1 (HIF-1). Surprisingly, I observed that COX-2 overexpression induced by hypoxia is always coupled to a significant decrease of miR-101(1) levels in colon cancer cell lines, suggesting that miR-101(1) regulation could be involved in the adaption of cancer cells to the hypoxic environment that strongly characterize CRC tissues.

Cáncer y tratamiento oncológico: Representaciones sociales de la población general, el paciente oncológico y miembros del equipo de salud

Se realizaron tres estudios cualitativos que tuvieron como propósito conocer las representaciones que ha construido la población general, los pacientes oncológicos y los profesionales de la salud, sobre el cáncer, la quimioterapia y el trasplante de médula ósea y realizar un análisis sobre las semejanzas y diferencias entre ellos. Se realizó en la ciudad de Bogotá (Colombia) con 55 personas: 20 pacientes con cáncer en proceso de trasplante de médula ósea, 20 personas no diagnosticadas con cáncer y 15 personas que trabajan en la atención de pacientes con cáncer. Se realizó una entrevista en profundidad con todos los participantes y asociaciones libres, clásicas y por sustitución sobre las palabras “cáncer”, “quimioterapia” y “trasplante de médula”. Los datos conseguidos se analizaron a la luz de la Teoría de las Representaciones Sociales (TRS). El análisis de la información siguió la técnica de análisis cualitativo de contenido para encontrar significados simbólicos y construir, denominar y definir categorías. Para los tres grupos el cáncer es una enfermedad terrible, que puede llevar a la muerte. El personal de salud y la población general creen que la enfermedad genera terror, angustia y miedo. Los pacientes tienen conciencia de la gravedad y del temor consecuente por una enfermedad que lo cambia todo, produce sufrimiento, dolor, obliga a depender de alguien y puede conducir a la muerte. El personal de salud considera que los pacientes lo pueden vivir como un castigo y la población general que puede ser la consecuencia de estilos de vida poco saludables. Para todos, la quimioterapia es un tratamiento para la enfermedad, que por un lado presenta efectos colaterales difíciles y visibles y que producen sentimientos negativos de temor y de angustia y al mismo tiempo constituye una opción y posibilidad de curación. El Trasplante de Médula Ósea representa para todos una oportunidad.

Strategies to hijack MTs dysfunction in neurodegenerative tauopathies: Design and synthesis of novel CNS-disease-modifying tools

Neuronal microtubules assembly and dynamics are regulated by several proteins including (MT)-associated protein tau, whose aberrant hyperphosphorylation promotes its dissociation from MTs and its abnormal deposition into neurofibrillary tangles, a common neurotoxic hallmarks of neurodegenerative tauopathies. To date, no disease-modifying drugs have been approved to combat CNS tau-related diseases. The multifactorial etiology of these conditions represents one of the major limits in the discovery of effective therapeutic options. In addition, tau protein functions are orchestrated by diverse post-translational modifications among which phosphorylation mediated by PKs plays a leading role. In this context, conventional single-target therapies are often inadequate in restoring perturbed networks and fraught with adverse side-effects. This thesis reports two distinct approaches to hijack MT defects in neurons. The first is focused on the rational design and synthesis of first-in-class triple inhibitors of GSK-3β, FYN, and DYRK1A, three close-related PKs, which act as master regulators of aberrant tau hyperphosphorylation. A merged multi-target pharmacophore strategy was applied to simultaneously modulate all three targets and achieve a disease-modifying effect. Optimization of ARN25068 by a computationally and crystallographic driven SAR exploration, allowed to rationalize the key structural modifications to maintain a balanced potency against all three targets and develop a new generation of quite well-balanced analogs exhibiting improved physicochemical properties, a good in vitro ADME profile, and promising cell-based anti-tau phosphorylation activity. In Part II, MT-stabilizing compounds have been developed to compensate MT defects in tau-related pathologies. Intensive chemical effort has been devoted to scaling up BL-0884, identified as a promising MT-normalizing TPD, which exhibited favorable ADME-PK, including brain penetration, oral bioavailability, and brain pharmacodynamic activity. A suitable functionalization of the exposed hydroxyl moiety of BL-0884 was carried out to generate corresponding esters and amides possessing a wide range of applications as prodrugs and active targeting for cancer chemotherapy.

Association between pre-treatment indicators and compliance to neoadjuvant/perioperative chemotherapy in operable gastric cancer

Background and aims: perioperative treatment is currently the gold standard approach for locally advanced gastric cancer (GC). Unfortunately, the phenomenon of patients dropping out of treatment has been frequently observed. The primary aims of this study were to verify if routine blood parameters, the inflammatory response markers, sarcopenia, and the depletion of adipose tissues were associated with compliance with neoadjuvant/perioperative chemotherapy. Methods and study design: sarcopenia and adipose indices were calculated with a CT scan before starting chemotherapy and before surgery. Blood samples were considered before the first and second cycles of chemotherapy. Results: A total of 84 patients with localized operable GC, were identified between September 2010 and January 2021. Forty-four patients (52.4%) did not complete the treatment according to the number of cycles planned/performed. Eight patients (9.5%) decided to suspend chemotherapy, seven patients (8.3%) discontinued because of clinical decision-making, 14 patients (16.7%) because of toxicity, and 15 patients (17.9%) for miscellaneous causes. Sarcopenia before starting chemotherapy was found to be present in 38 patients (50.7%) while it was in 47 patients (60%) at the CT scan before the gastrectomy. In multivariable analysis, both for changes tending to have a value of PLR at basal and in the second control a higher one than the cut-off (OR = 5.03, 95% CI: 1.34 - 18.89, p-value = 0.017), and for PLR which increased from a lower to a higher value in second control with respect to the cut off (OR = 4.64, 95% CI: 1.02 -21.02, p-value = 0.047) resulted associated with incomplete compliance. Conclusions: among the biological indicators, changes in the value of PLR with a tendency towards increasing compared to the cut-off appear to be an immediate indicator of incomplete compliance with neoadjuvant/perioperative treatment. More information is needed to reduce the causes of interruption.

Generation and characterization of mouse models of Small cell lung cancer and Basal cell carcinoma for the preclinical evaluation of new therapies

Background. Human small cell lung cancer (SCLC) accounting for approximately 15-20% of all lung cancers, is an aggressive tumor with high propensity for early regional and distant metastases. Although the initial tumor rate response to chemotherapy is very high, SCLC relapses after approximately 4 months in ED and 12 months in LD. Basal cell carcinoma (BCC) is the most prevalent cancer in the western world, and its incidence is increasing worldwide. This type of cancer rarely metastasizes and the death rate is extraordinary low. Surgery is curative for most of the patients, but for those that develop locally advanced or metastatic BCC there is currently no effective treatment. Both types of cancer have been deeply investigated and genetic alterations, MYCN amplification (MA) among the most interesting, have been found. These could become targets of new pharmacological therapies. Procedures. We created and characterized novel BLI xenograft orthotopic mouse models of SCLC to evaluate the tumor onset and progression and the efficacy of new pharmacological strategies. We compared an in vitro model with a transgenic mouse model of BCC, to investigate and delineate the canonical HH signalling pathway and its connections with other molecular pathways. Results and conclusions. The orthotopic models showed latency and progression patterns similar to human disease. Chemotherapy treatments improved survival rates and validated the in vivo model. The presence of MA and overexpression were confirmed in each model and we tested the efficacy of a new MYCN inhibitor in vitro. Preliminar data of BCC models highlighted Hedgehog pathway role and underlined the importance of both in vitro and in vivo strategies to achieve a better understanding of the pathology and to evaluate the applicability of new therapeutic compounds

Activity and mechanisms of action of novel organosulfur derivatives of the HDAC inhibitor Valproic Acid in human experimental models of non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) represents the leading cause of cancer death worldwide, and 5-year survival is about 16% for patients diagnosed with advanced lung cancer and about 70-90% when the disease is diagnosed and treated at earlier stages. Treatment of NSCLC is changed in the last years with the introduction of targeted agents, such as gefitinib and erlotinib, that have dramatically changed the natural history of NSCLC patients carrying specific mutations in the EGFR gene, or crizotinib, for patients with the EML4-ALK translocation. However, such patients represent only about 15-20% of all NSCLC patients, and for the remaining individuals conventional chemotherapy represents the standard choice yet, but response rate to thise type of treatment is only about 20%. Development of new drugs and new therapeutic approaches are so needed to improve patients outcome. In this project we aimed to analyse the antitumoral activity of two compounds with the ability to inhibit histone deacethylases (ACS 2 and ACS 33), derived from Valproic Acid and conjugated with H2S, in human cancer cell lines derived from NSCLC tissues. We showed that ACS 2 represents the more promising agent. It showed strong antitumoral and pro-apoptotic activities, by inducing membrane depolarization, cytocrome-c release and caspase 3 and 9 activation. It was able to reduce the invasive capacity of cells, through inhibition of metalloproteinases expression, and to induce a reduced chromatin condensation. This last characteristic is probably responsible for the observed high synergistic activity in combination with cisplatin. In conclusion our results highlight the potential role of the ACS 2 compound as new therapeutic option for NSCLC patients, especially in combination with cisplatin. If validated in in vivo models, this compound should be worthy for phase I clinical trials.

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.

CYP17A1 polymorphisms and clinical outcome of patients with metastatic castration-resistant prostate cancer treated with abiraterone

Background. Abiraterone acetate is a potent inhibitor of cytochrome P450 17 α-hydrolase (CYP17A1) that causes a reduction in the synthesis of testosterone in the adrenal glands, testes and tumor microenvironment. Blocking androgen production, abiraterone has been shown to prolong progression-free survival (PFS) and overall survival (OS) in patients with metastatic castration-resistant prostate cancer (CRPC) previously submitted to chemotherapy. The aim of our study was to verify the role of single nucleotide polymorphisms (SNPs) in predicting clinical outcome in CRPC patients treated with abiraterone after chemotherapy. Methods. We analyzed 48 CRPC consecutive patients treated with abiraterone after at least one chemotherapeutic regimen with docetaxel. DNA was extracted from peripheral blood and genotyped for four polymorphisms in the CYP17A1 gene (rs743572, rs10883783, rs17115100, rs284849). PFS and OS survival curves were used to identify statistical associations between haplotypes and clinical outcome. Results. Forty-eight Caucasian patients with metastatic CRPC treated with abiraterone were genotyped for polymorphisms in the CYP17A1 gene. All samples were evaluable for both sequencing and TaqMan Genotyping assay. The CRPC patients treated with abiraterone had a median PFS and OS of 7.6 months (95% CI: 4.3-10.5) and 17.6 months (95% CI: 10.5-19.0), respectively Statistical analyses highlighted a difference approaching statistical significance (log-rank test p = 0.0534) between rs10883783 and PFS. Other polymorphisms were not associated with a benefit from treatment with abiraterone. Conclusions. In our case series of 48 treated patients, rs10883783 only was identified as a possible predictive marker, results showing a trend toward statistical significance. Further analysis of this polymorphism is needed in larger series of patients to confirm our findings.

Outcome analysis of predictors in locally advanced pancreatic cancer

Guidelines report a wide range of options in locally advanced pancreatic cancer (LAPC): definitive chemotherapy or chemoradiotherapy or the emerging stereotactic body radiotherapy (SBRT) (+/- chemotherapy). On behalf of the AIRO (Italian Association of Radiation Oncology and Clinical Oncology) Gastrointestinal Study Group, we collected retrospective clinical data on 419 LAPC from 15 Italian centers. The study protocol (PAULA-1: Pooled Analysis in Unresectable Locally Advanced pancreatic cancer) was approved by institutional review board of S. Orsola-Malpighi Hospital (201/2015/O/OssN). From this large database we performed tree different studies. The first was a retrospective study about 56 LAPC treated with SBRT at a median biologically equivalent dose of 48 Gy +/- chemotherapy. We demonstrated a statistically significant impact of biologically equivalent dose based on an α/β ratio of 10Gy ≥ 48Gy for local control (LC) (p: 0.045) and overall survival (p: 0.042) in LAPC. The second was a retrospective matched-cohort case-control study comparing SBRT (40 patients) and chemoradiation (40 patients) in LAPC in terms of different endpoints. Our findings suggested an equivalence in terms of most outcomes among the two treatments and an advantage of SBRT in terms of LC (p: 0.017). The third study was a retrospective comparison of definitive chemotherapy, chemoradiotherapy and SBRT (+/- chemotherapy) in terms of different outcomes in LAPC. A predictive model for LC in LAPC was also developed reaching an AUC of 68% (CI 58,7%-77,4%). SBRT treatment emerged as a positive predictive factor for improved LC. Findings deriving from our three studies suggest that SBRT is comparable to standard of care (definitive chemotherapy and chemoradiotherapy) in terms of outcomes. SBRT seems to be an emerging therapeutic option in LAPC significantly improving local control. Furthermore, we have shown the potential of a predictive model for LC. Randomized trials are needed to compare these different therapeutic options in LAPC.

The human gut microbiome in disease: Role in chemotherapy treatments and relationship with clinical outcomes

The gut microbiome (GM) is a plastic entity, capable of adapting in response to intrinsic and extrinsic factors. However, several circumstances can disrupt this homeostatic balance, forcing the GM to shift from a health-associated mutualistic configuration to a disease-associated profile. Nowadays, a new frontier of microbiome research is understanding the GM role in chemo-immunotherapies and clinical outcomes. Here, the role of the genotoxin‐producing pathogen Salmonella in colorectal carcinogenesis was characterized by in-vitro models. A synergistic effect of Salmonella and the CRC-associated mutation (APC gene) promoted a tumorigenic microenvironment by increasing cellular genomic instability. Subsequently, the GM involvement in anti-cancer therapies was investigated via next-generation sequencing in different patient cohorts. The GM trajectory during treatments was characterized for women with epithelial ovarian cancer and pediatric patients undergoing hematopoietic stem cell transplantation (HSCT). The results highlighted the loss of GM homeostasis, with diversity reduction, decrease in health-associated microorganisms and pathobiont bloom. Interestingly, a distinctive GM profile was identified in ovarian cancer patients with a poor response to chemotherapy compared to patients in remission. Moreover, maintenance of GM homeostasis through enteral feeding in pediatric HSCT patients highlighted a better prognosis, with reduced risk of clinical complications. In this context, the gut resistome – the pattern of GM antibiotic-resistance genes (ARGs) – was evaluated longitudinally in HSCT patients. The results showed new acquisitions and consolidation of ARGs already present in patients developing clinical complications. Antibiotic exposure was also evaluated in infants under low-dose antibiotic prophylaxis for vesico-ureteral reflux showing an impairment of the GM configuration with possible long-term health implications. Dramatic GM dysbiosis was finally observed in critically ill patients with COVID-19 (undergoing multiple drug therapies) and correlated with increased risk of bloodstream infection. All these findings pointed out the importance of maintaining GM homeostasis during chemotherapy treatments for improving patients’ clinical outcomes.

A phase II, single arm study of CarbopLatin plus Etoposide with Bevacizumab and Atezolizumab in patients with exTEnded-disease small-cell lung cancer (SCLC) – CeLEBrATE trial

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor diagnosed at extended disease SCLC (ES-SCLC) stage in about 70% of cases. The new standard of treatment for patients with ES-SCLC is a combination of platinum-etoposide chemotherapy and atezolizumab or durvalumab, two programmed cell death ligand 1 (PD-L1) inhibitory monoclonal antibodies (mAb). However, the benefit derived from the addition of PD-L1 inhibitors to chemotherapy in ES-SCLC was limited and restricted to a subset of patients. The vascular endothelial growth factor (VEGF) is the most important pro-angiogenic factor implicated in cancer angiogenesis, which is abundant in SCLC and associated with poor prognosis. Antiangiogenic agents, such as bevacizumab, a humanized mAb against VEGF, added to platinum-etoposide chemotherapy improved progression-free survival in SCLC in two trials, but it did not translate into a benefit in overall survival. Nevertheless, VEGF has also acts as a mediator of an immunosuppressive microenvironment and its inhibition can revert the immune-suppressive tumor microenvironment and potentially enhance the efficacy of immunotherapies. Based on available preclinical data, we hypothesized that VEGF inhibition by bevacizumab could improve atezolizumab efficacy in a synergistic way and designed a phase II single-arm trial of bevacizumab in combination with carboplatin, etoposide, and atezolizumab as first-line treatment in ES-SCLC. The trial, which is still ongoing, enrolled 53 patients, including those with treated or untreated asymptomatic brain metastases (provided criteria are met), who received atezolizumab, bevacizumab, carboplatin and etoposide for 4-6 cycles (induction phase), followed by maintenance with atezolizumab and bevacizumab for a maximum of 18 total cycles or until disease progression, patient refusal, unacceptable toxicity. The evaluation of efficacy of the experimental combination in terms of 1-year overall survival rate is not yet mature (primary objective of the trial). The combination was feasible and the toxicity profile manageable (secondary objective of the trial).

Analysis of extracellular vesicles from patients with advanced pancreatic cancer identifies miRNAs with predictive value for treatment with gemcitabine + nab-paclitaxel

Pancreatic cancer (PC) is the seventh leading cause of cancer death. Despite recent therapy advancements, 5-year survival is 11%. Resistance to therapy is common, and no predictive factors, except for BRCA1/2 and PALB2 mutations, can drive treatment selection. Based on the easy isolation of extracellular vesicles (EVs) from blood and the role of EV-borne miRNAs in chemoresistance, we analyzed EVs and their miRNA content in order to identify predictive factors. First, we analyzed samples from 28 PC patients and 7 healthy subjects, in order to establish methods for isolation and analysis of EVs and their miRNA content. We observed a significantly different expression of 28 miRNAs, including oncogenic or tumor suppressor miRNAs, showing the ability of our approach to detect candidate biomarkers. Then, we analyzed samples of 21 advanced PC patients, collected before first-line treatment with gemcitabine + nab-paclitaxel, and compared findings in responders and non-responders. EVs have been analyzed with Nanoparticle tracking analysis, flow cytometry and RNA-Seq; then, laboratory results have been matched with clinical data. Nanoparticle tracking analysis did not show any significant difference. Flow cytometry showed a lower expression of SSE4 and CD81 in responders. Finally, miRNA analysis showed 25 upregulated and 19 downregulated miRNAs in responders. In particular, in responders we observed upregulation of miR-141-3p, miR-141-5p, miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-375-3p, miR-429, miR-545-5p. These miRNAs have targets with a previously reported role in PC. In conclusion, we show the feasibility of the proposed approach to identify EV-derived biomarkers with predictive value for therapy with gemcitabine + nab-paclitaxel in PC. Our findings highlight the possibility to exploit liquid biopsy for personalized treatment in PC, in order to maximize chances of response and patients’ outcome. These findings are worthy of further investigation: in the same setting, with different chemotherapy schedules, and in different disease settings such as preoperative therapy.

Understanding molecular mechanisms of resistance to immune checkpoint inhibitors in advanced non-small cell lung cancer (NSCLC)

Immune checkpoint inhibitors (ICI) that target PD-1/PD-L1 have recently emerged as an integral component of front-line treatment in metastatic NSCLC patients. The PD-1 inhibitor pembrolizumab is approved as monotherapy for advanced NSCLC with a PD-L1 tumor proportion score (TPS) of ≥1% and in combination with platinum doublet chemotherapy regardless of PD-L1 expression level. However, responses to either regimen occur in only a minority of cases, and PD-L1 TPS is limited as a biomarker in predicting whether a cancer will respond to PD-1 inhibition alone or would be more likely to benefit from PD-1 inhibition plus chemotherapy. Additional biomarkers of immunotherapy efficacy, such as tumor mutational burden (TMB), have not been incorporated into routine clinical practice for treatment selection. The identification of patients who have the greatest likelihood of responding to immunotherapies is critical for guiding treatment decisions. IN addition, early indicators of response could theoretically prevent patients from staying on an ineffective therapy where they might experience complications due to disease progression or develop toxicities from unnecessary exposure to an inactive agent. The aim of this research project is to investigate the clinicopathologic and molecular determinant of response/resistance to the currently available immune checkpoint inhibitors, in order to identify therapeutic vulnerabilities that can be exploited to improve the clinical outcomes of patients with advanced NSCLC.