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Lung cancer harbouring BRAF mutations accounts for 4% of all non-small cell lung cancer (NSCLC) cases, identifying a relevant subset of patients that need to be promptly managed. Three subtypes of BRAF mutations have been described: class I (V600E), and class II and III (non-V600), with different prognostic and predictive outcomes. Pivotal phase II trials have demonstrated the efficacy of the double BRAF/MEK inhibition with dabrafenib plus trametinib in patients harbouring V600E mutations, making BRAF a mandatory requirement in the genetic portrait of advanced non-squamous lung cancer patients. However, non-V600 mutations represent around 50% of BRAF-mutant NSCLC patients, for which no specific targeted approaches are approved. A paradigm shift from the double BRAF/MEK inhibition to combinations with agents with distinct mechanisms of action, such as immune-checkpoint inhibitors, pan-RAF and selective ERK 1/2 inhibitors, is under investigation and may change the therapeutic landscape of BRAF-driven NSCLC. This paper provides a practical, concise and updated review on the therapeutic strategies in NSCLC with BRAF mutations.

In NSCLC, KRAS mutations occur in up to 30% of all cases, most frequently at codon 12 and 13. KRAS mutations have been linked to adenocarcinoma histology, positive smoking history, and Caucasian ethnicity, although differences have been described across KRAS mutational variants subtypes. KRAS mutations often concur with other molecular alterations, notably TP53, STK11, and KEAP1, which could play an important role in treatment efficacy and patient outcomes. For many years, KRAS mutations have been considered undruggable mainly due to a high toxicity profile and low specificity of compounds. Sotorasib and adagrasib are novel KRAS inhibitors that recently gained FDA approval for pre-treated KRAS mutant NSCLC patients, and other molecules such as GDC-6036 are currently being investigated with promising results. Despite their approval, the efficacy of these drugs is lower than expected and progression among responders has been reported. Mechanisms of acquired resistance to anti-KRAS molecules typically involves either on target secondary mutations (e.g., G12, G13, Q61H, R68S, H95, Y96C, V8L) or off-target alterations. Ongoing trials are currently evaluating strategies for implementing efficacy and overcoming acquired resistance to these compounds. Finally, the efficacy of immune-checkpoint inhibitors still needs to be completely assessed and responses to anti-PD-1/PD-L1 agents may strongly depend on concomitant mutations.

RAF family proteins are serine–threonine kinases that play a central role in the MAPK pathway which is involved in embryogenesis, cell differentiation, cell proliferation and death. Deregulation of this pathway is found in up to 30% of all human cancers and BRAF mutations can be identified in 1.5–3.5% of NSCLC patients. Following the positive results obtained through the combination of BRAF and MEK inhibitors in BRAF-mutant melanoma, the same combination was prospectively assessed in BRAF-mutant NSCLC. In cohort B of the BRF113928 trial, 57 pretreated NSCLC patients were treated with dabrafenib plus trametinib: an ORR of 68.4%, a disease control rate of 80.7%, a median PFS of 10.2 months and a median OS of 18.2 months were observed. Similar results were reported in the first-line setting (cohort C), with an ORR of 63.9%, a DCR of 75% and a median PFS and OS of 10.2 and 17.3 months, respectively. The combination was well tolerated: the main adverse events were pyrexia (64%), nausea (56%), diarrhoea (56%), fatigue (36%), oedema (36%) and vomiting (33%). These positive results led to the approval of the combination of dabrafenib and trametinib for the treatment of BRAF V600E metastatic NSCLC patients regardless of previous therapy. Ongoing research should better define the role of new generation RAF inhibitors for patients with acquired resistance, the activity of chemo-immunotherapy or the combination of TKIs with chemotherapy or with immunotherapy in patients with BRAF-mutated cancers.

Aim: Comparison of first-line FOLFIRINOX (FFN) and nab-paclitaxel plus gemcitabine (NabGem) in patients with metastatic pancreatic ductal adenocarcinoma. Patients & methods: The authors analyzed data from 160 patients with metastatic pancreatic adenocarcinoma receiving first-line FFN (n=43) or NabGem (n=117). Results: FFN and NabGem were similar in median progression-free survival (24.43 vs 26.28weeks; hazard ratio [HR]: 0.88) and medial overall survival (47.43 vs 42.86weeks; HR: 0.90). Of the 43 patients receiving FFN, 26 (60.4%) were treated with second-line NabGem; 14/117 (12.0%) patients receiving NabGem received second-line FFN (p&nbsp

Background: CDK4/6 inhibitors have been approved in combination with endocrine therapy for the treatment of estrogen receptor positive (ER+) metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors. Results: p21-activated kinase 1 (PAK1) is a serine/threonine kinase amplified in several human cancer types. Specifically, the survey of TCGA for copy number alterations and/or expression showed PAK1 amplification and/or overexpression in ~23% of ER+ breast cancer. To test the hypothesis that PAK1-amplification is related to CDK4/6 inhibitors resistance, we generated two ER+ breast cancer cell lines T47D (harbouring PAK1 amplification) and MCF7 (non PAK1-amplified) resistant to the combination of fulvestrant (ER downregulator) and abemaciclib (CDK4/6 inhibitor), namely T47DFAR and MCF7FAR. T47DFAR and MCF7FAR were obtained, exposing the parental cells to increasing doses of drugs. Interestingly, only the T47DFAR but not the MCF7FAR showed higher level of PAK1 expression and activation compared to the parental cell line. PAK1 pathway activation was evaluated measuring by immunoblot the levels of phosphorylation of PAK1, MEK, ERK and Beta-Catenin. Moreover, the genomic knockdown of PAK1 through RNAi causes a decrease in cell proliferation of T47DFAR, but not of MCF7FAR and parental cells, suggesting the role of PAK1 as a possible mediator of drug resistance. Conversely, overexpression of PAK1 in MCF7 and T47D parental cells was able to generate resistance to the drug combination. PAK-1 has shown to have a different role in the cytosol as pathways mediator and in the nucleus as transcriptional factor. Our data obtained by subcellular fractionation showed that after treatment with fulvestrant-abemaciclib, PAK1 protein accumulates into the cytosol in T47D-FAR compared to parental T47D, probably mediating an enhancement in cytoplasmatic cell signalling and pathway activation. To univocally demonstrate that PAK1 mediates tumor resistance to fulvestrant-abemaciclib combination, we are generating data from RNA-Seq and whole exome sequencing from T47DFAR and MCF7FAR and the relative parental cell lines. Also, we will assess the efficacy of PAK pharmacological inhibition in overcoming such resistance. Conclusions: In conclusion, our data suggest the role of PAK1 as a novel therapeutic target in ER+/PAK1-amplified breast cancer model resistant to endocrine therapy and CDK4/6 inhibitors. Citation Format: Stefania Belli, Alberto Servetto, Concetta Di Mauro, Daniela Esposito, Ada Pesapane, Fabiana Napolitano, Antonio Santaniello, Pietro De Placido, Priscilla Cascetta, Annachiara Carratù, Eleonora Mozzillo, Roberta Marciano, Roberto Bianco, Luigi Formisano. Pak1 as a novel mediator of resistance to endocrine therapy and CDK4/6 inhibitors in ER+/PAK-1amplified breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-08.

Patients with locally advanced (LA) pancreatic ductal adenocarcinoma (PDAC) do not present distant metastases but are not eligible for surgery upfront. Chemotherapy regimens, such as FOLFIRINOX (FFN) or nab-paclitaxel plus gemcitabine (GemNab) in combination with loco-regional treatments are generally used in this setting. However, the best treatment choice is unknown. We retrospectively analyzed the information of 225 patients with stage II–III PDAC treated at our institution between October 2011 and December 2020. A total of 94 patients with LA PDAC who are non-eligible for surgery upfront received neoadjuvant FFN or GemNab. Of the 67 patients receiving FFN, 28 (41.8%) underwent surgery after neoadjuvant therapy. Of the 27 patients treated with GemNab, 6 (22.2%) became eligible for resection. The median overall survival (OS) was 85.1 weeks and 54.3 weeks in the FFN and GemNab groups, respectively (HR = 0.54, p = 0.0109). The median OS was 189.7 weeks and 76.4 weeks in the resected and unresected cohorts, respectively (HR = 0.25, p &lt, 0.0001). Neutropenia (37.3%), anemia (6.0%), and diarrhea (6.0%) in the FFN group and neutropenia (22.2%) and thrombocytopenia (18.5%) in the GemNab groups were the most frequent grade 3–4 side effects. Higher rates of thrombocytosis (p &lt, 0.0001) and peripheral edema (p &lt, 0.0001) were observed in the GemNab group. Our results suggest that the use of FFN is associated with more favorable clinical outcomes than GemNab for patients with LA PDAC. Future randomized and controlled clinical trials are needed to further elucidate the role of these regimens and loco-regional treatments in this setting.

Simple Summary Non-small cell lung cancer (NSCLC) remains a significant cause of death worldwide, despite the significant progresses to date. Multiple molecular alterations have been identified in NSCLC, leading to the development of target-based agents that have shown significant clinical benefits. Rearranged during Transfection (RET) fusions have recently emerged as a new potential target and a number of non-selective and selective RET inhibitors have been tested in RET positive NSCLC. In this review we analyse and summarise the characteristics of RET functions and its alterations in NSCLC. We then present the state of the art RET inhibitors in the treatment of NSCLC, discussing the ongoing trials and the future perspectives for RET positive (RET+) NSCLC patients. Abstract RET rearrangements are observed in 1–2% of non-small-cell lung cancer (NSCLC) patients and result in the constitutive activation of downstream pathways normally implied in cell proliferation, growth, differentiation and survival. In NSCLC patients, RET rearrangements have been associated with a history of non-smoking, a higher rate of brain metastasis at initial diagnosis and a low immune infiltrate. Traditionally, RET fusions are considered mutually exclusive with other oncogenic drivers, even though a co-occurrence with EGFR mutations and MET amplifications has been observed. Cabozantinib, vandetanib and lenvatinib are the first multi-kinase inhibitors tested in RET-rearranged NSCLC patients with contrasting results. More recently, two selective RET inhibitors, selpercatinib and pralsetinib, demonstrated higher efficacy rates and good tolerability and they were approved for the treatment of patients with metastatic RET fusion-positive NSCLC on the bases of the results of phase II studies. Two ongoing phase III clinical trials are currently comparing selpercatinib or pralsetinib to standard first line treatments and will definitively establish their efficacy in RET-positive NSCLC patients.

Immunotherapy has recently emerged as a novel strategy for treating different types of solid tumors, with promising results. However, still a large fraction of patients do not primarily respond to such approaches, and even responders sooner or later develop resistance. Moreover, immunotherapy is a promising strategy for certain malignancies but not for others, with this discrepancy having been attributed to a more immunogenic microenvironment of some tumors. As abnormal and augmented tumor vessels often occur in cancerogenesis, anti-angiogenic drugs have already demonstrated their effectiveness both in preclinical and in clinical settings. By targeting abnormal formation of tumor vessels, anti-angiogenetic agents potentially result in an enhanced infiltration of immune effector cells. Moreover, crosstalks downstream of the immune checkpoint axis and vascular endothelial growth factor receptor (VEGFR) signaling may result in synergistic effects of combined treatment in tumor cells. In this review, we will describe and discuss the biological rationale of a combined therapy, underlying the modification in tumor microenvironment as well as in tumor cells after exposure to checkpoint inhibitors and anti-angiogenic drugs. Moreover, we will highlight this strategy as a possible way for overcoming drug resistance. By first discussing potential prognostic and predictive factors for combined treatment, we will then turn to clinical settings, focusing on clinical trials where this strategy is currently being investigated.

Background: Pancreatic adenocarcinoma is the fourth leading cause of cancer-related death. In cases with metastasis, the combination of 5-fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX) or gemcitabine-based chemotherapy regimens are considered the standard of care. However, the optimal sequence of these regimens is unclear. Methods: This retrospective study initially evaluated 186 patients with locally advanced/metastatic pancreatic cancer at three Italian institutions between February 2013 and October 2019. All patients had progressed after receiving gemcitabine-based first-line chemotherapy and were subsequently offered second-line FOLFIRINOX, FOLFOX-6, or FOLFIRI treatment. This study evaluated progression-free survival (PFS), overall survival from the start of second-line treatment (OS2), overall survival from the start of first-line treatment (OS1), and safety outcomes. Results: A total of 77 patients received ⩾4 cycles of second-line chemotherapy and were considered eligible: 15 patients received FOLFIRINOX, 32 patients received FOLFOX-6, and 30 patients received FOLFIRI. The FOLFIRINOX group had median PFS of 26.29 weeks and median OS2 of 47.86 weeks, while the FOLFIRI group had median PFS of 10.57 weeks and median OS2 of 25.00 weeks ( p = 0.038). No significant differences were observed between the FOLFIRINOX and FOLFOX-6 groups in terms of PFS (26.29 weeks versus 23.07 weeks) or OS2 (47.86 weeks versus 42.00 weeks). The most common grade 3–4 toxicities were anemia, neutropenia, and thrombocytopenia, which occurred more frequently in the FOLFIRINOX and FOLFOX-6 groups. Conclusion: Relative to the FOLFIRI regimen, the FOLFIRINOX regimen had a favorable toxicity profile and better survival outcomes. No significant differences were observed relative to the FOLFOX-6 regimen.

The optimal therapeutic strategy for locally advanced pancreatic cancer patients (LAPC) has not yet been established. Our aim is to evaluate how surgery after neoadjuvant treatment with either FOLFIRINOX (FFN) or Gemcitabine-NabPaclitaxel (GemNab) affects the clinical outcome in these patients. LAPC patients treated at our institution were retrospectively analysed to reach this goal. The group characteristics were similar: 35 patients were treated with the FOLFIRINOX regimen and 21 patients with Gemcitabine Nab-Paclitaxel. The number of patients undergoing surgery was 14 in the FFN group (40%) and six in the GemNab group (28.6%). The median Disease-Free Survival (DFS) was 77.10 weeks in the FFN group and 58.65 weeks in the Gem Nab group (p = 0.625), while the median PFS in the unresected group was 49.4 weeks in the FFN group and 30.9 in the GemNab group (p = 0.0029, 95% CI 0.138&ndash, 0.862, HR 0.345). The overall survival (OS) in the resected population needs a longer follow up to be completely assessed, while the median overall survival (mOS) in the FFN group was 72.10 weeks and 53.30 weeks for the GemNab group (p = 0.06) in the unresected population. Surgery is a valuable option for LAPC patients and it is able to induce a relevant survival advantage. FOLFIRINOX and Gem-NabPaclitaxel should be offered as first options to pancreatic cancer patients in the locally advanced setting.

Background: Recently, immune check-point inhibitors have shown efficacy in triple-negative breast cancer (TNBC). Since Hedgehog (Hh) signalling mediates crosstalk between breast cancer cells and tumor-infiltrating immune cells, we investigated the mechanisms by which Hh can modulate PDL1 expression and the tumor-immune microenvironment. Methods: TNBC tumors from untreated patients were subjects to PDL1 and Gli1 expression analysis by IHC. The correlation of Hh pathway activation and PDL1 expression was assessed in TNBC cells treated with the SMO-inhibitor NVP-LDE225. The main aim was to study the PDL1/Gli1 cross-talk. Results: The expression of PDL1 and Gli1, the major indicator for the canonical Hh signaling activation, were assessed by IHC in a tissue microarray (TMA) of TNBC samples from 237 untreated patients. In 203/237 cases we could analyze both PDL1 and Gli1 protein expression. A significant correlation between PDL1 and Gli1 expression was found. Indeed, of 77/203 (38%) PDL1 positive tumors 42/77 (54%) expressed Gli1. In order to explore correlations between other Hh pathway members and PDL1 in TNBCs, we interrogated the open-access database TCGA; PDL1 positive TNBCs showed high levels of SMO and PTCH1, Hh pathway receptors (Q-value 0.018 and 0.010). To better understand the link between Hh pathway and PDL1, we selected a panel of TNBC cell lines; the pharmacological Hh pathway inhibition led to a reduction of PDL1 protein and mRNA. On the other hand, engineered cells harbouring Gli1 overexpression showed higher levels of PDL1. Therefore, we hypothesized that Hh pathway could be involved in the PDL1 transcriptional modulation. To address this issue, we performed a luciferase reporter assay and a ChIP analysis following by PCR amplification of the PDL1 gene promoter. We found that Gli1 binds the PDL1 promoter, indicating that Gli1 transcriptionally enhances PDL1 expression. Gli1 expression was evaluated also in 4T1 cells, a TNBC murine model; furthermore, in 4T1 cells PDL1 protein expression was inhibited by NVP-LDE225. In vivo experiment will be performed in Balb/C mice orthotopically xenografted with 4T1 cells to confirm the role of Hh pathway to modulate the PDL1 expression and the tumor microenviroment in vivo. Conclusions: Our results suggest that Hh pathway has a crucial role in cancer immune evasion trough PDL-1 modulation. Due to their ability to target both tumor cells and the tumor microenvironment, Hh inhibitors could represent promising therapeutics to be clinically investigated in Gli1 overexpressing TNBC patients. Citation Format: Pietro De Placido, Concetta Di Mauro, Daniela Esposito, Ada Pesapane, Stefania Belli, Fabiana Napolitano, Antonio Santaniello, Priscilla Cascetta, Annachiara Carratù, Eleonora Mozzillo, Roberta Marciano, Alberto Servetto, Roberto Bianco, Luigi Formisano. Hedgehog pathway is involved in cancer immune surveillance through PDL1 modulation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-17.

Cancer and obesity are the two major epidemics of the 21st century. Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of death, with a five-year overall survival rate of only 8%. Its incidence and mortality have increased in recent years, and this cancer type is expected to be among the top five leading causes of cancer-related death by 2030 in the United States (US). In the last three decades, the prevalence of overweight people has boosted with a consequent increase in obesity-related diseases. Considerable epidemiologic evidence correlates overweight and obese conditions to an increased risk of several types of cancer, including PDAC. Besides being a risk factor for multiple metabolic disorders, the tumor-promoting effects of obesity occur at the local level via inflammatory mediators that are associated with adipose inflammation and metabolic or hormones mediators and microbiota dysbiosis. Although an excess of body mass index (BMI) represents the second most modifiable risk factor for PDAC with an increased cancer related-death of more than 20–40%, still little is known about the molecular mechanisms that underlie this strong association. In this review, we focused on the role of obesity as a preventable risk factor of PDAC, discussing the molecular mechanisms linking obesity to cancer initiation and progression. Moreover, we highlighted the role of obesity in defining chemoresistance, showing how a high BMI can actually reduce response to chemotherapy.

Inhibition of the mechanistic target of rapamycin (mTOR) is a promising treatment strategy for several cancer types. Rapamycin derivatives such as everolimus are allosteric mTOR inhibitors acting through interaction with the intracellular immunophilin FKBP12, a prolyl isomerase with different cellular functions. Although mTOR inhibitors have significantly improved survival of different cancer patients, resistance and lack of predictive factors of response remain unsolved issues. To elucidate the mechanisms of resistance to everolimus, we evaluated Met activation in everolimus-sensitive/resistant human cancer cells, in vitro and in vivo. Biochemical and computational analyses were performed. Everolimus-resistant cells were xenografted into mice (10/group) and studied for their response to everolimus and Met inhibitors. The statistical significance of the in vitro results was evaluated by Student's t test. Everolimus reduced Met phosphorylation in everolimus-sensitive cells. This event was mediated by the formation of a Met-FKBP12 complex, which in turn is disrupted by everolimus. Aberrant Met activation in everolimus-resistant cells and overexpression of wild-type/mutant Met caused everolimus resistance. Pharmacological inhibition and RNA silencing of Met are effective in condition of everolimus resistance (P