PO-473 Quantification of ERCC1-XPF complexes in ovarian cancer xenografts with different sensitivity to cisplatin

Introduction Epithelial ovarian cancer is the most lethal gynaecological cancer due to the development of resistance to a platinum based therapy. As DNA repair capacity is a key determinant for the cellular response to platinum (DDP) agents, DNA repair functional assays are required to study its relevance in DDP resistance. We set up a proximity ligation assay (PLA) to study the activity of nucleotide excision repair (NER) in patient derived ovarian carcinoma xenografts (PDXs) sensitive (S) and resistant (R) to DDP. Material and methods Patient derived xenografts from fresh ovarian carcinomas were recently established in our laboratory. DDP antitumour activity was evaluated in most of the PDXs. Mice were sacrificed when tumours reached 1,5–2 gr. Tumours were fixed in formalin and paraffin embedded (FFPE). PLA was performed on tumour slides, using DuolinkII reagents (Sigma-Aldrich) and following the manufacturer instructions. PLA detects the presence of the protein complexes ERCC1-XPF, that are quantified as foci per nucleus and represent a biomarker of NER activity. Images were acquired by Olympus Virtual Slider (Olympus) and analysed with ImageJ software. Statistical analysis was performed with GraphPad Prism7. Results and discussions Our xenobank comprises PDXs with different response to DDP: MNHOC266 and MNHOC230 are very sensitive to the drug, while MNHOC315 is resistant. We also obtained three sublines resistant to DDP (MNHOC124R, MNHOC124LPR and MNHOC239R) starting from sensitive PDXs (MNHOC124S, MNHOC124LPS and MNHOC239S), after several in vivo drug treatments. Statistically significant higher level of ERCC1-XPF foci could be observed in MNHOC124R and MNHOC124LPR as compared to their sensitive counterparts. No differences were observed between MNHOC239S and R PDXs, even if the number of ERCC1-XPF foci in MNHOC239S were statistically higher than the ones observed in MNHOC124S and in MNHOC124LPS. MNHOC266 and MNHOC230 showed levels of foci comparable to those of MNHOC124S and MNHOC124LPS. mRNA and protein levels of the different isoforms of ERCC1 and of XPF were not different among the PDXs studied. Conclusion PLA for the detection of ERCC1-XPF complexes was set up in FFPE xenograft tumour slides. These preliminary results highlight a possible link between DDP resistance and higher NER activity that need to be confirmed in a wider panel of PDXs. In addition, these data confirm the importance to develop functional assays to directly evaluate the activity of different DNA repair pathways to predict DDP activity

Association between Antibiotic-Immunotherapy Exposure Ratio and outcome in metastatic Non Small Cell Lung Cancer

OBJECTIVES: Immunotherapy (IO) is effective in metastatic Non Small Cell Lung Cancer (NSCLC). Gut microbiota has an impact on immunity and its imbalance due to antibiotics may impair the efficacy of IO. We investigated this topic in a case series of NSCLC patients treated with IO. MATERIALS AND METHODS: Data about all metastatic NSCLC patients treated with IO between 04/2013 and 01/2018 were collected. Patients were stratified according to antibiotic use during the Early IO Period (EIOP), and according to the Antibiotic-ImmunotherapyExposure Ratio (AIER) defined as "days of antibiotic/days of IO" during the Whole IO Period (WIOP). Survival was estimated using the Kaplan-Meier method. Log-rank test was used to compare the curves. Multivariate analyses were performed with the Cox model. RESULTS: We analyzed 157 patients. Forty-six patients received antibiotics during the WIOP, 27 patients during the EIOP. No differences in either Progression-Free Survival (PFS) or Overall Survival (OS) were observed according to antibiotic use during the EIOP (p\u2009=\u20090.1772 and p\u2009=\u20090.2492, respectively). Considering the WIOP, median AIER was 4.2%. The patients with a higher AIER had worse PFS (p\u2009<\u20090.0001) and OS (p\u2009=\u20090.0004) than the others. Results were significant also after correction for the IO line (p\u2009=\u20090.0018 for PFS) and performance status (p\u2009<\u20090.0001 for PFS, p\u2009=\u20090.0052 for OS). CONCLUSION: Although no difference in outcome were observed with antibiotic use in the EIOP, a detrimental effect became evident for patients with a higher AIER in the WIOP. If its relevance is confirmed, AIER may become an innovative variable for estimating the impact of antibiotics on IO efficacy

Choosing wisely first line immunotherapy in non-small cell lung cancer (NSCLC): what to add and what to leave out

Immunotherapy has dramatically changed the therapeutic scenario in treatment na\uefve advanced non-small celllung cancer (NSCLC). While single agent pembrolizumab has become the standard therapy in patients with PD-L1 expression on tumor cells 65 50%, the combination of pembrolizumab or atezolizumab and platinum-basedchemotherapy has emerged as an effective first line treatment regardless of PD-L1 expression both in squamousand non-squamous NSCLC without oncogenic drivers. Furthermore, double immune checkpoint inhibition hasshown promising results in treatment na\uefve patients with high tumor mutational burden (TMB). Of note, thepresence of both negative PD-L1 expression and low TMB may identify a subgroup of patients who has littlebenefit from immunotherapy combinations and for whom the best treatment option may still be platinum-basedchemotherapy. To date, first-line single agent immune checkpoint blockade has demonstrated limited activity inEGFR mutated NSCLC and the combination of immunotherapy and targeted agents has raised safety concerns inboth EGFR and ALK positive NSCLC patients. Finally, in EGFR mutated or ALK rearranged NSCLC, atezolizumabin combination with platinum-based chemotherapy and bevacizumab is emerging as a potential treatment optionupon progression to first line tyrosine kinase inhibitors

Efficacy and safety of immunotherapy in elderly patients with non-small cell lung cancer

OBJECTIVES: Most trials with Immune Checkpoint Inhibitors (ICIs) for Non-Small Cell Lung Cancer (NSCLC) included only small subgroups of patients aged 6565. As NSCLC is often diagnosed in patients aged 6570, real-world data about efficacy and safety of immunotherapy (IO) in elderly patients are essential. MATERIALS AND METHODS: We retrospectively collected data about all patients with advanced NSCLC treated with IO at our Institution between April 2013 and March 2019. The patients were stratified for age as follows: <70 year-old, 70-79 year-old, 6580 year-old. Chi-square test was used to compare qualitative variables. Survival was estimated with Kaplan-Meier method. Log-rank test was used to compare curves. Multivariate analyses were performed with Cox model. RESULTS: We reviewed 290 cases, with a median age of 67 (range: 29-89). Patients aged<70, 70-79 and 6580 year-old were 180, 94 and 16, respectively. Clinical/pathological variables were uniformly distributed across age classes, except for a higher rate of males (p 0.0228) and squamous histology (p 0.0071) in the intermediate class. Response Rate (RR) was similar across age groups (p 0.9470). Median Progression Free Survival (PFS) and Overall Survival (OS) did not differ according to age (p 0.2020 and 0.9144, respectively). Toxicity was comparable across subgroups (p 0.6493). The only variables influencing outcome were performance status (PS) (p\u2009<\u20090.0001 for PFS, p 0.0192 for OS), number of metastatic sites (p 0.0842 for PFS, p 0.0235 for OS) and IO line (p\u2009<\u20090.0001 for both PFS and OS). CONCLUSION: Advanced age was not associated to a reduced efficacy of IO in our case series. Furthermore, no toxicity concern emerged even among the eldest pts. To our opinion, ICIs should be considered irrespective of age, provided an optimal PS at baseline. Of note, IO is often the only therapeutic option applicable to these cases considering the toxicity of chemotherapy

Single-arm, open label prospective trial to assess prediction of the role of ERCC1/XPF complex in the response of advanced NSCLC patients to platinum-based chemotherapy

Background: Platinum-based therapy, combined or not with immune checkpoint inhibitors, represents a front-line choice for patients with non-small-cell lung cancer (NSCLC). Despite the improved outcomes in the last years for this malignancy, only a sub-group of patients have long-term benefit. Excision repair cross-complementation group 1 (ERCC1) has been considered a potential biomarker to predict the outcome of platinum-based chemotherapy in NSCLC. However, the ERCC1 gene is transcribed in four splice variants where the isoform 202 was described as the only one active and able to complex Xeroderma pigmentosum group F-complementing protein (XPF). Here, we prospectively investigated if the active form of ERCC1, as assessed by the ERCC1/XPF complex (ERCC1/XPF), could predict the sensitivity to platinum compounds.Patients and methods: Prospectively enrolled, patients with advanced NSCLC treated with a first-line regimen containing platinum were centrally evaluated for ERCC1/XPF by a proximity ligation assay. Overall survival (OS), progression-free survival (PFS) and objective response rate (ORR) were analyzed.Results: The absence of the ERCC1/XPF in the tumor suggested a trend of worst outcomes in terms of both OS [hazard ratio (HR) 1.41, 95% confidence interval (CI) 0.67-2.94, P 0.373] and PFS (HR 1.61, 95% CI 0.88-3.03, P = 0.123). ORR was marginally influenced in ERCC1/XPF-negative and -positive groups [odds ratio (stable disease progressive disease versus complete response partial response) 0.87, 95% a 0.25-3.07, P = 0.832].Conclusion: The lack of ERCC1/XPF complex in NSCLC tumor cells might delineate a group of patients with poor outcomes when treated with platinum compounds. ERCC1/XPF absence might well identify patients for whom a different therapeutic approach could be necessary

Characterization of a new trabectedin-resistant myxoid liposarcoma cell line that shows collateral sensitivity to methylating agents

Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and β, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin

SMO mutations confer poor prognosis in malignant pleural mesothelioma

Background: Malignant pleural mesothelioma (MPM) is an aggressive tumor but approximately 12% of patients survive more than 3 years. The biological differences underlying better outcomes are not known. Several targeted agents and immunotherapy have been ineffective. Hedgehog (Hh) is one emerging pathway. We compared the biological profiles of patients with different survival, investigating the most frequently altered genes, including the Hh pathway. Methods: We analyzed 56 MPM. A 36-month overall survival (OS) cut-off divided patients into 32 normo (NS) and 24 long (LS) survivors. We used next generation sequencing to test 21 genes, immunohistochemistry to evaluate SMO expression. Mutation differences between NS and LS and their associations with clinical features were analysed by Fisher's test, OS with the Kaplan-Meier method and its association with mutations by univariate and multivariate Cox proportional hazard models. Results: Clinical features were similar in both groups. Eighteen out of 56 patients (32%) were wild-type for the genes analysed. At least five had mutations in BAP1, NF2, TP53, SMO and PTCH1 with no significant differences between the groups except for SMO. SMO, a member of the Hh pathway, was mutated only in NS (15.6%) and only SMO mutations were significantly associated with poor prognosis at univariate (HR =4.36, 95% CI: 2.32-8.18, P<0.0001) and multivariate (HR =9.2, 95% CI: 3.0-28.4, P=0.0001) analysis. All SMO mutated patients expressed high protein levels. Conclusions: SMO mutations were clearly associated with worse prognosis. SMO may be a therapeutic target but this needs to be confirmed in a prospective trial

Artificial intelligence for predictive biomarker discovery in immuno-oncology: a systematic review

Background: The widespread use of immune checkpoint inhibitors (ICIs) has revolutionised treatment of multiple cancer types. However, selecting patients who may benefit from ICI remains challenging. Artificial intelligence (AI) approaches allow exploitation of high-dimension oncological data in research and development of precision immuno-oncology. Materials and methods: We conducted a systematic literature review of peer-reviewed original articles studying the ICI efficacy prediction in cancer patients across five data modalities: genomics (including genomics, transcriptomics, and epigenomics), radiomics, digital pathology (pathomics), and real-world and multimodality data. Results: A total of 90 studies were included in this systematic review, with 80% published in 2021-2022. Among them, 37 studies included genomic, 20 radiomic, 8 pathomic, 20 real-world, and 5 multimodal data. Standard machine learning (ML) methods were used in 72% of studies, deep learning (DL) methods in 22%, and both in 6%. The most frequently studied cancer type was non-small-cell lung cancer (36%), followed by melanoma (16%), while 25% included pan-cancer studies. No prospective study design incorporated AI-based methodologies from the outset; rather, all implemented AI as a post hoc analysis. Novel biomarkers for ICI in radiomics and pathomics were identified using AI approaches, and molecular biomarkers have expanded past genomics into transcriptomics and epigenomics. Finally, complex algorithms and new types of AI-based markers, such as meta-biomarkers, are emerging by integrating multimodal/multi-omics data. Conclusion: AI-based methods have expanded the horizon for biomarker discovery, demonstrating the power of integrating multimodal data from existing datasets to discover new meta-biomarkers. While most of the included studies showed promise for AI-based prediction of benefit from immunotherapy, none provided high-level evidence for immediate practice change. A priori planned prospective trial designs are needed to cover all lifecycle steps of these software biomarkers, from development and validation to integration into clinical practice

Modulation of peripheral blood immune cells by early use of steroids and its association with clinical outcomes in patients with metastatic non-small cell lung cancer treated with immune checkpoint inhibitors

Background Steroids are frequently used in patients with metastatic non-small cell lung cancer (mNSCLC), but they could be detrimental for patients treated with immune checkpoint inhibitors (ICIs). Here, we assessed the association between early use of steroids, clinical outcomes and peripheral immune blood cells modulation in patients with mNSCLC treated with ICIs. Methods We reviewed patients with mNSCLC treated at our institution between April 2013 and December 2017. Early use of steroids was defined as the use of a daily prednisone-equivalent dose >= 10 mg for at least 1 day within 28 days after ICI initiation. Peripheral immune blood cell counts were retrieved at baseline and at 4 and 6 weeks after ICI initiation. Results Out of 151 patients included, 35 (23%) made early use of steroids that was associated with poor disease control (OR 0.32, p=0.006), progression-free survival (HR 1.80, p=0.003) and overall survival (HR 2.60, p<0.001). Early use of steroids significantly correlated with higher median absolute neutrophil count, neutrophil to lymphocyte ratio (NLR) and derived NLR, and lower median absolute and relative eosinophil count, both at 4 and 6 weeks after ICI initiation. Conclusions In patients with mNSCLC treated with ICIs, early use of steroids was associated with worse clinical outcomes and remarkable modulation of peripheral blood immune cells, which could contribute to restraining the activation of antitumour immunity. If confirmed in prospective studies, these findings would highlight the importance of carefully evaluating and, whenever possible, avoiding steroids during early phases of ICI treatment

Metformin Enhances Cisplatin-Induced Apoptosis and Prevents Resistance to Cisplatin in Co-mutated KRAS/LKB1 NSCLC

Introduction: We hypothesized that activating KRAS mutations and inactivation of the liver kinase B1 (LKB1) oncosuppressor can cooperate to sustain NSCLC aggressiveness. We also hypothesized that the growth advantage of KRAS/LKB1 co-mutated tumors could be balanced by higher sensitivity to metabolic stress conditions, such as metformin treatment, thus revealing new strategies to target this aggressive NSCLC subtype. Methods: We retrospectively determined the frequency and prognostic value of KRAS/LKB1 co-mutations in tissue specimens from NSCLC patients enrolled in the TAILOR trial. We generated stable LKB1 knockdown and LKB1-overexpressing isogenic H1299 and A549 cell variants, respectively, to test the in vitro efficacy of metformin. We also investigated the effect of metformin on cisplatin-resistant CD133+cells in NSCLC patient-derived xenografts. Results: We found a trend towards worse overall survival in patients with KRAS/LKB1 co-mutated tumors as compared to KRAS-mutated ones (hazard ratio: 2.02, 95% confidence interval: 0.94\u20134.35, p = 0.072). In preclinical experiments, metformin produced pro-apoptotic effects and enhanced cisplatin anticancer activity specifically in KRAS/LKB1 co-mutated patient-derived xenografts. Moreover, metformin prevented the development of acquired tumor resistance to 5 consecutive cycles of cisplatin treatment (75% response rate with metformin-cisplatin as compared to 0% response rate with cisplatin), while reducing CD133+cells. Conclusions: LKB1 mutations, especially when combined with KRAS mutations, may define a specific and more aggressive NSCLC subtype. Metformin synergizes with cisplatin against KRAS/LKB1 co-mutated tumors, and may prevent or delay the onset of resistance to cisplatin by targeting CD133+cancer stem cells. This study lays the foundations for combining metformin with standard platinum-based chemotherapy in the treatment of KRAS/LKB1 co-mutated NSCLC