Synergic activity of FGFR2 and MEK inhibitors in the treatment of FGFR2-amplified cancers of unknown primary.

Patients with cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies. Experimental models are pivotal for CUP biology investigation and drug testing. We derived two CUP cell lines (CUP#55 and #96) and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines and PDXs underwent histological, immune-phenotypical, molecular, and genomic characterization confirming the features of the original tumor. The tissue-of-origin prediction was obtained from the tumor microRNA expression profile and confirmed by single-cell transcriptomics. Genomic testing and fluorescence in situ hybridization analysis identified FGFR2 gene amplification in both models, in the form of homogeneously staining region (HSR) in CUP#55 and double minutes in CUP#96. FGFR2 was recognized as the main oncogenic driver and therapeutic target. FGFR2-targeting drug BGJ398 (infigratinib) in combination with the MEK inhibitor trametinib proved to be synergic and exceptionally active, both in vitro and in vivo. The effects of the combined treatment by single-cell gene expression analysis revealed a remarkable plasticity of tumor cells and the greater sensitivity of cells with epithelial phenotype. This study brings personalized therapy closer to CUP patients and provides the rationale for FGFR2 and MEK targeting in metastatic tumors with FGFR2 pathway activation.

Automated Prediction of the Response to Neoadjuvant Chemoradiotherapy in Patients Affected by Rectal Cancer.

BACKGROUND: Rectal cancer is a malignant neoplasm of the large intestine resulting from the uncontrolled proliferation of the rectal tract. Predicting the pathologic response of neoadjuvant chemoradiotherapy at an MRI primary staging scan in patients affected by locally advanced rectal cancer (LARC) could lead to significant improvement in the survival and quality of life of the patients. In this study, the possibility of automatizing this estimation from a primary staging MRI scan, using a fully automated artificial intelligence-based model for the segmentation and consequent characterization of the tumor areas using radiomic features was evaluated. The TRG score was used to evaluate the clinical outcome. METHODS: Forty-three patients under treatment in the IRCCS Sant'Orsola-Malpighi Polyclinic were retrospectively selected for the study; a U-Net model was trained for the automated segmentation of the tumor areas; the radiomic features were collected and used to predict the tumor regression grade (TRG) score. RESULTS: The segmentation of tumor areas outperformed the state-of-the-art results in terms of the Dice score coefficient or was comparable to them but with the advantage of considering mucinous cases. Analysis of the radiomic features extracted from the lesion areas allowed us to predict the TRG score, with the results agreeing with the state-of-the-art results. CONCLUSIONS: The results obtained regarding TRG prediction using the proposed fully automated pipeline prove its possible usage as a viable decision support system for radiologists in clinical practice.

The autocrine loop of ALK receptor and ALKAL2 ligand is an actionable target in consensus molecular subtype 1 colon cancer.

BACKGROUND: In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs). METHODS: In this work, we performed a meta-analysis of CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested cell lines, patient-derived organoids and mice with potent ALK inhibitors, already approved for clinical use. RESULTS: ALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D tumor spheroids. Consistently, ALK appeares constitutively phosphorylated in CMS1, and it signals mainly through the AKT axis. Mechanistically, we found that CMS1 cells display several copies of ALKAL2 ligand and ALK-mRNAs, suggesting an autocrine loop mediated by ALKAL2 in the activation of ALK pathway, responsible for the invasive phenotype. Consequently, disruption of ALK axis mediates the pro-apoptotic action of CMS1 cell lines, both in 2D and 3D and enhanced cell-cell adhesion and e-cadherin organization. In agreement with all these findings, the ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, as a proof of concept, the efficacy of ALK inhibition was demonstrated in both patient-derived organoids and in tumor xenografts in vivo. CONCLUSIONS: Collectively, these findings suggest that ALK targeting may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC.

Unexpected response to fourth-line paclitaxel in a patient with metastatic oropharyngeal squamous cell carcinoma, immunotherapy-refractory: a case report.

In recent years, immune checkpoint inhibitors (ICIs), including nivolumab and pembrolizumab have revolutionized the treatment landscape in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). However, many patients do not respond to ICIs for reasons that remain largely unknown. For patients who progress on ICIs, chemotherapy and/or biologic therapies are the most widely used treatments based on the clinician's choice, with no defined sequence strategy. We report the experience of a patient with metastatic oropharyngeal squamous cell cancer p16 and human papillomavirus-DNA positive who received chemotherapy with weekly paclitaxel after progressing on nivolumab. Our patient presented a partial response to fourth line paclitaxel, which lasted more than 2 years, with an improvement of his quality of life too. These results support the hypothesis of synergism between immunotherapy and conventional chemotherapies. Even in the setting of immune-refractory disease, immunotherapy may affect tumor immune microenvironment thus leading to a synergistic effect with conventional chemotherapy and achieving unexpected results.

A phase II, open-label, single-arm trial of carboplatin plus etoposide with bevacizumab and atezolizumab in patients with extended-stage small-cell lung cancer (CeLEBrATE study): background, design and rationale.

Based on improved survival from the addition of PD-L1 inhibitors in phase III trials, the combination of immunotherapy and platinum-doublet chemotherapy has become the new standard treatment for extended-stage small-cell lung cancer (ES-SCLC). Furthermore, the antiangiogenetic agent bevacizumab showed a longer progression-free survival by targeting VEGF that has pleiotropic effects, including immunosuppressive ones. We, therefore, hypothesized that targeting angiogenesis would improve the efficacy of chemoimmunotherapy. The CeLEBrATE trial is an open-label, multicenter, phase II study designed to assess the efficacy and safety of the combination of carboplatin and etoposide plus bevacizumab and atezolizumab in treatment-naive patients with ES-SCLC. The primary end point is overall survival rate at 1 year, while secondary end points include overall response rate, progression-free survival and toxicity.

Lay abstract Extended-stage small-cell lung cancer (ES-SCLC) is a highly aggressive lung cancer subtype, accounting for 13­15% of all lung cancers. For several years, the standard treatment for this disease was based on polychemotherapy, with a rapid disease response but with an equally rapid disease progression. The new standard treatment has recently been changed, based on the results of two large clinical trials, which showed the efficacy and safety of the combination of chemotherapy with immunotherapy compared to chemotherapy alone. Nevertheless, prognosis of ES-SCLC remains poor, and new treatment strategies are urgently needed. Therefore, we designed the CeLEBrATE trial to investigate whether the combination of chemotherapy with antiangiogenetic therapy and immunotherapy is safe and could improve survival in patients with ES-SCLC.

Novel HER2-Directed Treatments in Advanced Gastric Carcinoma: AnotHER Paradigm Shift?

Human epidermal growth factor receptor 2 (HER2) is overexpressed and/or amplified in approximately 15-20% of gastric adenocarcinoma (GC) patients. In 2010, the landmark ToGA trial established the combination of trastuzumab plus chemotherapy as the first-line standard of care for HER2-positive GC patients with advanced disease. However, subsequent studies on HER2 targeted therapies in this setting failed to meet their primary endpoints, and not all HER2-positive GC patients benefit from targeted approaches. More recently, novel HER2-directed treatments have been investigated, including trastuzumab deruxtecan (T-Dxd); following the results of the DESTINY-Gastric01 study, T-Dxd received its first U.S. Food and Drug Administration (FDA) approval on 15 January 2021 for the treatment of adults with unresectable, locally advanced, or metastatic GC who have received a prior trastuzumab-based regimen. In this review, we discuss the current HER2-targeted treatments for GC in the advanced disease setting, mainly focusing on emerging new treatments and future research directions.

Tumor-Associated Macrophages and Inflammatory Microenvironment in Gastric Cancer: Novel Translational Implications.

Gastric cancer (GC) represents the fifth most frequently diagnosed cancer worldwide, with a poor prognosis in patients with advanced disease despite many improvements in systemic treatments in the last decade. In fact, GC has shown resistance to several treatment options, and thus, notable efforts have been focused on the research and identification of novel therapeutic targets in this setting. The tumor microenvironment (TME) has emerged as a potential therapeutic target in several malignancies including GC, due to its pivotal role in cancer progression and drug resistance. Therefore, several agents and therapeutic strategies targeting the TME are currently under assessment in both preclinical and clinical studies. The present study provides an overview of available evidence of the inflammatory TME in GC, highlighting different types of tumor-associated cells and implications for future therapeutic strategies.

Expected and non-expected immune-related adverse events detectable by CT.

PURPOSE: Cancer treatments with immune checkpoint inhibitors (ICI) are associated with a unique set of drug toxicities called immune-related adverse events (irAES). The aim of the present study was to describe the radiological manifestation of irAES detectable by CT. METHOD: Retrospective analysis of 284 patients treated with ICI for various types of advanced cancer; of them, 129 patients were selected, all having been treated with single-agent ICI, and all with a baseline CT scan and follow-up scans available at our Institute. CT examinations were reviewed by two radiologists involved in the study with a consensus reading. Imaging findings consistent with irAES were reported and correlated with clinical-laboratory data. RESULTS: Immune-related adverse events were found in 25/129 (19.4 %) patients. No statistically significant differences were found in either the prevalence of irAES or in the time of onset of tumour type. Thoracic complications were detected in 14/25 (56.0 %) patients consisting in: 3 radiation recall pneumonia, 3 Transient Asymptomatic Pulmonary Opacities (TAPOs), 3 hypersensitivity pneumonia, 2 diffuse alveolar damage, 2 organizing pneumonia, 1 sarcoid-like reaction. In the remaining 11/25 (44.0 %), there were extra-pulmonary complications: 3 colitis, 4 cholecystitis, 2 pancreatitis and 2 cases of visceral ischemia. CONCLUSIONS: Radiologists should be aware of the wide spectrum of irAES as they could affect the outcome. Pneumonia is the most frequent irAES; however, the international classification for interstitial lung disease does not seem to be capable of describing all possible drug-related pulmonary toxicities. Additional findings included TAPOs, radiation recall pneumonia and sarcoid-like reaction.

Reliability of programmed death ligand 1 (PD-L1) tumor proportion score (TPS) on cytological smears in advanced non-small cell lung cancer: a prospective validation study.

INTRODUCTION: Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) assessment is mandatory for the single agent pembrolizumab treatment of patients with advanced non-small cell lung cancer (NSCLC). PD-L1 testing has been validated and is currently certified only on formalin-fixed paraffin-embedded materials but not on cytological smears. Unfortunately, a significant proportion of patients, having only cytological material available, cannot be tested for PD-L1 and treated with pembrolizumab. In this study, we aimed to validate PD-L1 IHC on cytological smears prospectively by comparing clone SP263 staining in 150 paired histological samples and cytological smears of NSCLC patients. METHODS: We prospectively enrolled 150 consecutive advanced NSCLC patients. The clone SP263 was selected as, in a previous study of our group, it showed higher accuracy compared with clones 28-8 and 22-C3, with good cyto-histological agreement using a cut-off of 50%. For cyto-histological concordance, we calculated the kappa coefficient using two different cut-offs according to the percentage of PD-L1 positive neoplastic cells (<1%, 1-49% and ⩾50%; <50%, ⩾50%). RESULTS: The overall agreement between histological samples and cytological smears was moderate (kappa = 0.537). However, when the cyto-histological concordance was calculated using the cut-off of 50%, the agreement was good (kappa = 0.740). With the same cut-off, and assuming as gold-standard the results on formalin-fixed paraffin-embedded materials, PD-L1 evaluation on smears showed specificity and negative predictive values of 98.1% and 93.9%, respectively. CONCLUSION: Cytological smears can be used in routine clinical practice for PD-L1 assessment with a cut-off of 50%, expanding the potential pool of NSCLC patients as candidates for first-line single agent pembrolizumab therapy.

Roles of IL-1 in Cancer: From Tumor Progression to Resistance to Targeted Therapies.

IL-1 belongs to a family of 11 members and is one of the seven receptor-agonists with pro-inflammatory activity. Beyond its biological role as a regulator of the innate immune response, IL-1 is involved in stress and chronic inflammation, therefore it is responsible for several pathological conditions. In particular, IL-1 is known to exert a critical function in malignancies, influencing the tumor microenvironment and promoting cancer initiation and progression. Thus, it orchestrates immunosuppression recruiting pro-tumor immune cells of myeloid origin. Furthermore, new recent findings showed that this cytokine can be directly produced by tumor cells in a positive feedback loop and contributes to the failure of targeted therapy. Activation of anti-apoptotic signaling pathways and senescence are some of the mechanisms recently proposed, but the role of IL-1 in tumor cells refractory to standard therapies needs to be further investigated.

Two first-in-human studies of xentuzumab, a humanised insulin-like growth factor (IGF)-neutralising antibody, in patients with advanced solid tumours.

BACKGROUND: Xentuzumab, an insulin-like growth factor (IGF)-1/IGF-2-neutralising antibody, binds IGF-1 and IGF-2, inhibiting their growth-promoting signalling. Two first-in-human trials assessed the maximum-tolerated/relevant biological dose (MTD/RBD), safety, pharmacokinetics, pharmacodynamics, and activity of xentuzumab in advanced/metastatic solid cancers. METHODS: These phase 1, open-label trials comprised dose-finding (part I; 3 + 3 design) and expansion cohorts (part II; selected tumours; RBD [weekly dosing]). Primary endpoints were MTD/RBD. RESULTS: Study 1280.1 involved 61 patients (part I: xentuzumab 10-1800 mg weekly, n = 48; part II: 1000 mg weekly, n = 13); study 1280.2, 64 patients (part I: 10-3600 mg three-weekly, n = 33; part II: 1000 mg weekly, n = 31). One dose-limiting toxicity occurred; the MTD was not reached for either schedule. Adverse events were generally grade 1/2, mostly gastrointestinal. Xentuzumab showed dose-proportional pharmacokinetics. Total plasma IGF-1 increased dose dependently, plateauing at ~1000 mg/week; at ≥450 mg/week, IGF bioactivity was almost undetectable. Two partial responses occurred (poorly differentiated nasopharyngeal carcinoma and peripheral primitive neuroectodermal tumour). Integration of biomarker and response data by Bayesian Logistic Regression Modeling (BLRM) confirmed the RBD. CONCLUSIONS: Xentuzumab was well tolerated; MTD was not reached. RBD was 1000 mg weekly, confirmed by BLRM. Xentuzumab showed preliminary anti-tumour activity. CLINICAL TRIAL REGISTRATION: NCT01403974; NCT01317420.

Emerging therapies in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare cancer of the pleural surfaces frequently related to asbestos exposure. It is characterized by a poor prognosis even for patients treated with trimodality therapy, including surgery, chemotherapy and radiotherapy. Moreover, the majority of patients are not candidates for surgery due to disease advanced stage or medical comorbidities. For these patients, the survival rate is even lower and few therapeutic options are currently available. Nevertheless, many interesting novel approaches are under investigation, among which immunotherapy represents one of the most promising emerging strategies. In this review, we will discuss the role of new therapeutic options, particularly immunotherapy, and present the results of the most important and promising clinical trials.

MYC Amplification as a Potential Mechanism of Primary Resistance to Crizotinib in ALK-Rearranged Non-Small Cell Lung Cancer: A Brief Report.

INTRODUCTION: Translocations of the anaplastic lymphoma kinase (ALK) can be effectively targeted in advanced non-small cell lung cancer by ALK-TKI inhibitors including Crizotinib. However, the development of acquired resistance often limits the duration of these therapies. While several mechanisms of secondary resistance have been already identified, little is known about molecular determinants of primary resistance. In our brief report we investigated the tumor molecular profile of a patient who failed to respond to Crizotinib. METHODS: Fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) were run on tumor specimen as well as search and characterization of circulating tumor cells (CTCs) in the blood. Confirmation of clinical findings was achieved using a translational cell-line in vitro model. RESULTS: We identified the amplification of MYC as a potential new mechanism of primary resistance to ALK inhibition. Human EML4-ALK rearranged cells infected with a lentiviral vector carrying full-length human MYC cDNA were treated in vitro with crizotinib and alectinib. Overexpression of MYC overexpression was associated with a reduced sensitivity to both ALK-inhibitors. MYC-overexpressing clones displayed also increased levels of both cyclin D and E and their growth was reduced by using Cdk4/6 inhibitors such as Palbociclib. CONCLUSIONS: We postulate that the MYC gene may be implicated in the mechanism of primary resistance to ALK inhibitors. We also suggest potential MYC-directed inhibition strategies to overcome primary resistance in advanced ALK-rearranged NSCLC.

Safety, efficacy and survival of patients with primary malignant brain tumours (PMBT) in phase I (Ph1) trials: the 12-year Royal Marsden experience.

BACKGROUND: Primary malignant brain tumours (PMBT) constitute less than 2% of all malignancies and carry a dismal prognosis. Treatment options at relapse are limited. First-in-human solid tumour studies have historically excluded patients with PMBT due to the poor prognosis, concomitant drug interactions and concerns regarding toxicities. METHODS: Retrospective data were collected on clinical and tumour characteristics of patients referred for consideration of Ph1 trials in the Royal Marsden Hospital between June 2004 and August 2016. Survival analyses were performed using the Kaplan-Meier method, Cox proportional hazards model. Chi squared test was used to measure bivariate associations between categorical variables. RESULTS: 100pts with advanced PMBT were referred. At initial consultation, patients had a median ECOG PS 1, median age 48 years (range 18-70); 69% were men, 76% had glioblastoma; 68% were on AEDs, 63% required steroid therapy; median number of prior treatments was two. Median OS for patients treated on a Ph1 trials was 9.3 months (95% CI 5.9-12.9) versus 5.3 months (95% CI 4.1-6.1) for patients that did not proceed with a Ph1 trial, p = 0.0094. Steroid use, poor PS, neutrophil-to-lymphocyte ratio and treatment on a Ph1 trial were shown to independently influence OS. CONCLUSIONS: We report a survival benefit for patients with PMBT treated on Ph1 trials. Toxicity and efficacy outcomes were comparable to the general Ph1 population. In the absence of an internationally recognized standard second line treatment for patients with recurrent PMBT, more Ph1 trials should allow enrolment of patients with refractory PMBT and Ph1 trial participation should be considered at an earlier stage.

Chemotherapy treatment in malignant pleural mesothelioma: a difficult history.

Malignant pleural mesothelioma (MPM) is a rare neoplasm that typically arises from mesothelial surfaces of the pleural cavity. Despite treatment improvements, it carries a dismal prognosis. The majority of patients either have unresectable disease or are not candidates for surgery due to medical comorbidities or old age. For such patients, chemotherapy (CT) represents the gold-standard treatment. To date, combination CT with cisplatin plus pemetrexed represents the most widely used regimen in first-line setting for patients with unresectable MPM. Other first-line options are currently available, including the use of raltitrexed instead of pemetrexed combined with platinum. In this review, we discuss the role of CT in MPM mainly focusing on the results of the trials conducted in first-line setting.

A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products.

Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the Ampli1™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.

Determinants of Last-line Treatment in Metastatic Breast Cancer.

BACKGROUND: In metastatic breast cancer (MBC) patients, the identification of factors helping clinicians in the choice between active therapy versus best supportive care is needed clinically. The aim of the present study was to identify the clinicopathologic factors that could improve the prognostic valuation of MBC patients and clinical decision-making at the end of life. PATIENTS AND METHODS: The present study analyzed data from a retrospective series of 522 MBC patients treated at the oncology department (University Hospital of Udine) from January 2004 to June 2014. The association between clinicopathologic features and death within 30 or 90 days since last-line treatment prescription was explored. Differences between lightly (≤ 3 lines) and heavily (> 3 lines) pretreated patients and the factors affecting treatment choice were investigated. RESULTS: The event "death" occurred in 410 patients. The median last-line survival was 100 days. The median number of therapeutic lines was 3. On multivariate analysis, worse Eastern Cooperative Oncology Group performance status was significantly associated with death within 90 and 30 days since last-line treatment prescription. Among the heavily pretreated patients, liver function impairment and evaluation by a breast cancer specialist were significantly associated with a greater and lower risk of death within 30 days, respectively. Among the lightly pretreated patients with luminal disease, age < 70 years, luminal B-like disease, and number of previous lines were associated with a greater chance of receiving chemotherapy. CONCLUSION: In the present study, the Eastern Cooperative Oncology Group performance status was the most robust independent factor driving the last-line therapeutic choice for MBC patients. In addition, the molecular subtype and oncologist subspecialization also influenced the decision-making process.