No abstract

No abstract

Resistance to Immune Checkpoint Blockade (ICB) constitutes the current limiting factor for the optimal implementation of this novel therapy, which otherwise demonstrates durable responses with acceptable toxicity scores. This limitation is exacerbated by a lack of robust biomarkers. In this study, we have dissected the basal TME composition at the gene expression and cellular levels that predict response to Nivolumab and prognosis. BCR, TCR and HLA profiling were employed for further characterization of the molecular variables associated with response. The findings were validated using a single-cell RNA-seq data of metastatic melanoma patients treated with ICB, and by multispectral immunofluorescence. Finally, machine learning was employed to construct a prediction algorithm that was validated across eight metastatic melanoma cohorts treated with ICB. Using this strategy, we have unmasked a major role played by basal intratumoral Plasma cells expressing high levels of IGKC in efficacy. IGKC, differentially expressed in good responders, was also identified within the Top response-related BCR clonotypes, together with IGK variants. These results were validated at gene, cellular and protein levels; CD138+ Plasma-like and Plasma cells were more abundant in good responders and correlated with the same RNA-seq-defined fraction. Finally, we generated a 15-gene prediction model that outperformed the current reference score in eight ICB-treated metastatic melanoma cohorts. The evidenced major contribution of basal intratumoral IGKC and Plasma cells in good response and outcome in ICB in metastatic melanoma is a groundbreaking finding in the field beyond the role of T lymphocytes.

Targeted therapy in metastatic melanoma often achieves a major tumour regression response and significant long-term survival via the release of antigens that reinduce immunocompetence. The biomarkers thus activated may guide the prediction of response, but this association and its mechanism have yet to be established. Blood samples were collected from nineteen consecutive patients with metastatic melanoma before, during, and after treatment with targeted therapy. Differential gene expression analysis was performed, which identified the genes involved in the treatment, both in the first evaluation of response and during progression. Although clinical characteristics of the patients were poorer than those obtained in pivotal studies, radiological responses were similar to those reported previously (objective response rate: 73.7%). In the first tumour assessment, the expression of some genes increased (CXCL-10, SERPING1, PDL1, and PDL2), while that of others decreased (ARG1, IL18R1, IL18RAP, IL1R1, ILR2, FLT3, SLC11A1, CD163, and S100A12). The analysis of gene expression in blood shows that some are activated and others inhibited by targeted therapy. This response pattern may provide biomarkers of the immune reinduction response, which could be used to study potential combination treatments. Nevertheless, further studies are needed to validate these results.

Methods: CRISP (NCT02622581) is a prospective, observational, open, multicentre, interdisciplinary clinical research platform that collects data on all (sequential) treatments, patient and tumour chr, biomarker testing, clinical and patient-reported out in w180 hospitals and practices in Germany. Currently 6300+ pts were recruited, who will be followed until death or end of project. Data from 493 pts with PD-L1 TPS50% recruited between 12/2015 and 06/2019 and receiving CPI as 1 st -line treatment was analysed. Pts were deemed se if they had the following chr: ECOG¼0-1, Stage IV, no brain metastases, no HIV or second tumour and no prior (neo-)adjuvant therapies.Results: Of 473 analysed pts 191 pts were potentially se in reflection of inclusion criteria for clinical trials KEYNOTE 42 and 24, 282 pts were n-se representing the realworld pts population. Although both groups are similar in age (median 68 years, both) and CR/PR rates were comparable (1%[se] vs. 2%[n-se] and 21% vs. 22%) as well as rates of stopped treatments because of toxicity (10%, both), potentially se pts. had a longer PFS (10.

No abstract

Melanoma comprises a variety of malignant cell types from the skin, the mucous membranes, and the pigmented cells of the eye. Its incidence and mortality rates continue to rise worldwide, mainly driven by increased ultraviolet light exposure. Although screening improvements have led to a better diagnosis in the early stages, the outcome for patients with Metastatic Melanoma (MM) remains poor and the survival rate is considerably low. Recently, Immune Checkpoint Inhibitors (ICB) have revolutionized the treatment of MM, although only 30% of patients enjoy clinical benefit and a plethora of different molecular mechanisms is postulated to cause resistance. Several biomarkers are currently on the spotlight as putative predictors of response; however, they are not sufficiently informative and robust. Circular RNAs (circRNAs) are a class of single-stranded stable RNAs produced by 5′-to-3′ transcription of coding gene exons or long non-coding RNAs that act as translation templates, RNA-binding protein regulators, or miRNA-binding sponges. Here, we present an exploratory study to evaluate the potential use of circRNA as ICB response predictors. We recruited 23 MM patients treated with Nivolumab and categorized them as good responders (10 patients) and bad responders (11 patients). All RNA-seq with ribosomal depletion was performed on RNA isolated from pre-treatment FFPE tumor biopsies. Two bioinformatic tools that use de novo assembly for the identification of circRNAs (Starchip and Ciri) were employed independently to characterize the MM circRNAs profile and to identify a specific pattern for responders. Overall, a total of 731 circRNAs were detected. Interestingly, the number of circRNAs were comparatively higher in bad responders (11.917 reads, 321 circRNAs) versus good responders (7368 reads, 176 circRNAs), with 234 common circRNAs. Additionally, we also explored the biological function of the identified circRNAs in MM, by interrogating KEGG, GO and Reactome databases to delineate the gene regulatory networks specifically associated with the response-related phenotypes. The circRNAs exclusively expressed in patients presenting resistance to Nivolumab were associated with Transcriptional Misregulation in Cancer and Viral Infections processes. In contrast, circRNAs expressed uniquely in good responders were enriched in Generic Transcription pathways. Importantly, amongst the identified circRNAs, CDR1-AS was differentially downregulated in good responders (536 vs 1580 reads, p-value 0.002). Remarkably, CDR1-AS acts as miRNA-binding sponge of miRNA-7 in the context of invasion and migration in melanoma. In conclusion, our exploratory study paves the way to the utilization of novel biomarkers associated to immunotherapy failure in MM patients. Moreover, our results suggest that CDR1-AS is a novel putative predictor of ICB response. Citation Format: Juan Luis Onieva, Javier Oliver, Aurora Laborda-Illanes, Maria Rosario Chica-Parrado, Alicia Garrido-Aranda, Cynthia Robles-Podadera, Daniel Prieto, Elena Gallego, Alfonso Sanchez, Iñaki Comino, Vanessa De Luque, Martina Alvarez, Maria Jose Lozano, Pedro Jimenez, Miguel Angel Berciano-Guerrero, Emilio Alba, Manuel Cobo, Isabel Barragan. Evaluation of circular RNA profiling in metastatic melanoma patients treated with immune checkpoint blockade [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 281.

Cutaneous melanoma (CM) is an aggressive malignant tumor of epidermal melanocytes for which treatment options and survival chances decrease dramatically when metastatic. To date, immunotherapy is becoming a new standard treatment for CM although less than 30% of the patients´ response positively. Therefore, there is a strong interest to identify response predictors and molecular pathways that might lead to therapy failure. Two novel biomarker sources are LncRNA and CircRNA which can function as natural sponges of miRNA as competitive endogenous RNAs (ceRNA) or can associate to RNA-Binding Proteins (RBP) to regulate gene expression. Moreover, several studies have revealed that ceRNA networks are implicated in tumorigenic processes. Considering this, we set to interrogate the role of the ceRNA in the prediction of response to CM immunotherapy. In this pilot study, differential expression was quantified by RNA-seq in 16 FFPE pre-treatment metastatic samples from CM patients treated with Nivolumab. Total RNA isolation was performed from 10 µm slides (4) with RNAeasy FFPE kit. Total RNAseq was done with Truseq Stranded RNA gold kit, and libraries were sequenced on Illumina Nextseq 550. LncRNA were obtained by STARs alignment and explored on LncATLAS. To assess the presence of high confident cirRNA we used 5 different pipelines with a minimum filtering cut-off of 2 junction reads in at least 2 samples and with at least 3 software. Deseq2 pipeline of total mapped reads was used to perform differential expression. We identified 23 circRNA and 74 lncRNA differentially expressed (DE) with a fold change of 1.5 and a p value < 0,01. Ingenuity pathway analysis was carried out to generate DE ceRNA-mRNA networks and associated RBP networks. Interestingly, in the DE ceRNA-mRNA networks, most of the 47 significant canonical pathways comprised immunological pathways such the antigen presentation pathway, Th1 and Th2 activation pathways and PD1-PDL1 cancer immunotherapy pathways. Regarding the DE ceRNA associated RBP networks, HOTAIR regulatory pathway and senescence as well as G1/S cell cycle checkpoint regulator pathways were enriched. Furthermore, we were able to predict the interaction of the DE ceRNA with the activation of key promoters of the immune response such as IL27 (Interleukin 27), EIB3 (Epstein-Barr Virus Induced 3), IL2 (Interleukin 2) and IFNA2 (Interferon Alpha2), as well as the inhibition of negative immune regulators such as SAFB2 (Distal-Less Homeobox 2) and FOXD1 (Forkhead Box D1). In conclusion, we have evidenced for the first time the potential utility of ceRNA as immunotherapy predictors. We have generated a response signature of ceRNA expression and used network analysis to associate them with specific key modulators of the immune response. This study opens a new venue for the investigation of the role of these regulatory ncRNA in the resistance to immunotherapy in cancer. Citation Format: Javier Oliver, Juan Luis Onieva, Maria Garrido-Barros, Alicia Garrido-Aranda, Vanessa De Luque, Martina Alvarez, Alfonso Sanchez, Elisabeth Perez, Patricia Chaves, Maria Jose Lozano, Miguel Berciano, Manolo Cobo, Emilio Alba, Antonio Rueda, Isabel Barragan. Association of ceRNA dysregulation with clinical Response to Immunotherapy in cutaneous melanoma (CM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1538.