Abstract Background Prognostic factors for initial response of advanced cutaneous squamous cell carcinoma to cemiplimab treatment are lacking. Il-6 has been found to affect immune cell populations which impact tumor development. The aim was to investigate the prognostic significance of IL-6 serum levels before and during treatment. Methods Serum levels of IL-6 were correlated with clinical outcomes in a retrospective study. Results Overall, 39 patients were enrolled. High serum levels of IL-6 (> 5.6 pg/ml) were associated with poorer survival (45.1% vs 0 deaths; OS: 16.1 ± 1.5 vs 20.8 ± 0 months, 95% CI 13,046 to 19,184) and shorter PFS (10.3 ± 1.9 vs 18.9 ± 1.5 months; 95% CI 3433 to 10,133) in patients with advanced CSCC treated with cemiplimab. In addition, patients whose IL-6 level increased after treatment with cemiplimab, independently of the basal level, had a poorer response to treatment than patients whose level was reduced or stable after immunotherapy. Conclusions Serum levels of IL-6 at baseline and changes after cemiplimab immunotherapy may have a prognostic significance in patients with advanced cutaneous squamous cell carcinoma.
The availability of immune checkpoint inhibitors (ICI) has radically changed the prognosis of patients (pts) with metastatic melanoma. More recently, PD-1 inhibitors have also become the standard of care as adjuvant therapy in high-risk and resected melanoma.1–7 However, 65–80% of patients treated with ICI experience immune-related adverse events (irAEs)8 causing morbidity and a potential decrease in clinical benefit. Currently, there are no biomarkers that can predict which patients are at risk of developing irAEs. Aim of this study is to recognize patients who will develop toxicity to anti-PD1 treatment.
Methods
Gene profiling analysis was performed using NanoString IO360 panel from basal PBMCs of patients treated with anti-PD1 in both setting (adjuvant and first line therapy). Patient's characteristics are reported in (table 1). To identify the best genes signature the Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) was applied. Differences in characteristics of patients with and without toxicity were tested by t-test or Wilcoxon test (according to their distribution) and Pearson chi-squared test for continuous and categorical variables, respectively. ROC curves were used to determine the best cut off which defines the border line between absence and presence of toxicity (table 2).
Results
Among 161 pts included, 75 received anti-PD1 as adjuvant therapy (AT) and 86 as first line therapy (FLT). Arthralgia and fever were observed in the 27% and 14% of total population (12% and 9% respectively in AT; 15% and 4% respectively in FLT). A specific gene signature for predicting the onset of arthralgia has been identified and characterized by the most representative genes such as: ZEB2, TNFS13, RPTOR, NFKBIE, GNLY, CCNB1. In particular, we observed that genes involved in the mTORC/NF-κB pathway correlate with arthralgia. Differently, the gene signature for predicting the onset of fever is mainly characterized by humoral immunity genes such as: HLA-F, CD8B, CD45RA, CD27. In both cases, the increase of signature value is associated with a greater probability of toxicity onset.
Conclusions
In this retrospective study, we found a gene signature model able to predict the onset of toxicities (arthralgia and fever) related to anti-PD1 treatment. Further investigations are needed to get additional information.
References
Carlino MS, Larkin J, Long GV. Immune checkpoint inhibitors in melanoma. Lancet. 2021;398(10304):1002–14. https://doi.org/10.1016/S0140–6736(21)01206-X. Jiang YZ, Liu YR, Xu XE, Jin X, Hu X, Yu KD, Shao ZM. Transcriptome analysis of triple-negative breast cancer reveals an integrated mRNA-lncRNA signature with predictive and prognostic value. Cancer Res. 2016;76(8):2105–14. https://doi.org/10.1158/0008–5472.CAN-15–3284. Gandhi S, Pandey MR, Attwood K, Ji W, Witkiewicz AK, Knudsen ES, Allen C, Tario JD, Wallace PK, Cedeno CD, Levis M, Stack S, Funchain P, Drabick JJ, Bucsek MJ, Puzanov I, 2021 John SP, Sun J, Carlson RJ, Cao B, Bradfield CJ, Song J, Smelkinson M, Fraser IDC. IFIT1 exerts opposing regulatory effects on the inflammatory and interferon gene programs in LPS-activated human macrophages. Cell Rep. 2018;25(1):95–106.e6. https://doi.org/10.1016/j.celrep.2018.09.002. Larkin J, Hodi FS, Wolchok JD. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med. 2015;373(13):1270–1. https://doi.org/10.1056/NEJMc1509660. Li JW, Zhou J, Shi ZT, Li N, Zhou SC, Chang C. Sonographic features of triple-negative breast carcinomas are correlated with mRNA-lncRNA signatures and risk of tumor recurrence. Front Oncol. 2021;10: 587422. https://doi.org/10.3389/fonc.2020.587422. Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000 Prime Rep. 2014;6:13. https://doi.org/10.12703/P6–13. Bertrand A, Kostine M, Barnetche T, Truchetet ME, Schaeverbeke T. Immune related adverse events associated with anti-CTLA-4 antibodies: systematic review and meta-analysis. BMC Med. 2015 Sep 4;13:211. doi: 10.1186/s12916–015-0455–8.
Ethics Approval
This study was approved by the Ethics Committee of Istituto Nazionale Tumori - IRCCS - Fondazione 'G. Pascale', Naples, Italy, protocol number 33/17 oss. All patients provided their written informed consent to participate in this study.
Consent
Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal
Monoclonal antibodies targeting immune checkpoints improved clinical outcome of patients with malignant melanoma. However, the mechanisms are not fully elucidated. Since immune check-point receptors are also expressed by helper innate lymphoid cells (ILCs), we investigated the capability of immune checkpoints inhibitors to modulate ILCs in metastatic melanoma patients as well as melanoma cells effects on ILC functions. Here, we demonstrated that, compared to healthy donors, patients showed a higher frequency of total peripheral ILCs, lower percentages of CD117+ ILC2s and CD117+ ILCs as well as higher frequencies of CD117- ILCs. Functionally, melanoma patients also displayed an impaired TNFα secretion by CD117- ILCs and CD117+ ILCs. Nivolumab therapy reduced the frequency of total peripheral ILCs but increased the percentage of CD117- ILC2s and enhanced the capability of ILC2s and CD117+ ILCs to secrete IL-13 and TNFα, respectively. Before Nivolumab therapy, high CCL2 serum levels were associated with longer Overall Survival and Progression Free Survival. After two months of treatment, CD117- ILC2s frequency as well as serum concentrations of IL-6, CXCL8 and VEGF negatively correlated with both the parameters. Moreover, melanoma cells boosted TNFα production in all ILC subsets and increased the number of IL-13 producing ILC2s in vitro. Our work shows for the first time that PD-1 blockade is able to affect ILCs proportions and functions in melanoma patients and that a specific subpopulation is associated with the therapy response.
The real-life application of immune checkpoint inhibitors (ICIs) may yield different outcomes compared to the benefit presented in clinical trials. For this reason, there is a need to define the group of patients that may benefit from treatment. We retrospectively investigated 578 metastatic melanoma patients treated with ICIs at the Istituto Nazionale Tumori IRCCS Fondazione "G. Pascale" of Napoli, Italy (INT-NA). To compare patients' clinical variables (i.e., age, lactate dehydrogenase (LDH), neutrophil-lymphocyte ratio (NLR), eosinophil, BRAF status, previous treatment) and their predictive and prognostic power in a comprehensive, non-hierarchical manner, a clinical categorization algorithm (CLICAL) was defined and validated by the application of a machine learning algorithm-survival random forest (SRF-CLICAL). The comprehensive analysis of the clinical parameters by log risk-based algorithms resulted in predictive signatures that could identify groups of patients with great benefit or not, regardless of the ICI received. From a real-life retrospective analysis of metastatic melanoma patients, we generated and validated an algorithm based on machine learning that could assist with the clinical decision of whether or not to apply ICI therapy by defining five signatures of predictability with 95% accuracy.
The immune checkpoint inhibitors revolutioned cancer therapeutic landscape and substantially improved the survival of patients with advanced malignancies, especially in skin cancer patients.1–5 Several predictive biomarkers are under evaluation, in order to identify patients who can derive benefit from ICI while also limiting exposure and toxicity. IL-6 is a pleiotropic cytokine involved not only in immune responses but it is also a major player in chronic inflammatory diseases.6–7 Additionally, elevated levels of IL-6 are observed in a large number of patients (pts) with solid tumours.8 The purpose of this study is to retrospectively investigate the relationships between IL-6 serum concentration and outcome in skin cancer patients treated with immunotherapy.
Methods
From June 2020 to October 2021 at INT IRCCS Pascale, Naples, we analyzed interleukin -6 from 265 consecutive serum samples in different skin cancer pts before and during immunotherapy treatment. We included pts with cutaneous squamous cell carcinoma (SCC) treated with cemiplimab (n=32), melanoma in adjuvant setting (n=61), metastatic melanoma treated with: anti-PD1 (n=103), combo ipi+nivo (n=36) and ipilimumab alone (n=32). All patients signed informed consent. Patients baseline characteristics are listed in table 1. IL6 ere measured by Electrochemiluminescence immunoassays (ECLIA) from Roche Cobas. ROC curves were used to determine the best cut off. Survival rates were analyzed using the Kaplan-Meier method and differences among curves were assessed by the log-rank test. Hazard Ratios (HR) and their 95% confidence intervals (CI) were estimated using a Cox regression model.
Results
Among 265 pts, lower serum concentration of interleukin-6 was associated with a better PFS (15.07 months (95% CI 9,76 to 18,86) versus 8.01 months (95% CI 2,80 to 4,03), HR = 0.34 (CI 0,23–0,50, p<0.0001), OS (19.83 months (95% CI 18.57 to 21.03) versus 14.53 months (95% CI 12.38 to 16.68), HR = 0.41 (CI 0,26–0,64, p=0.0001) and ORR (95% CI 6.86 to 13.30, p<0.001). Similarly, IL6 (p <0.01) and ORR (p <0.01) are significantly associated with OS and PFS in the multivariate analysis. We also confirmed the association between IL6 with PFS and OS in adjuvant setting. Moreover, pts with an IL-6 ratio (on treatment/baseline) ≤1 have a better PFS and OS.
Conclusions
In this retrospective study, we found that lower IL-6 level are associated with better OS, PFS and ORR. In addition, minimum variation of IL-6 during immunotherapy are strongly associated to outcome. Further investigations are needed to get additional information.
Acknowledgements
We thank the Italian Ministry of Health (IT-MOH) through support of 'Ricerca Corrente'
References
James Larkin, Vanna Chiarion-Sileni, Rene Gonzalez, Jean Jacques Grob, C. Lance Cowey et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015 Jul 2;373(1):23–34. Robert C, Long GV, Brady B, Dutriaux C, Maio M et al. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med. 2015 Jan 22;372(4):320–30. Weber JS, D'Angelo SP, Minor D, Hodi FS, Gutzmer R. Nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4 treatment (CheckMate 037): a randomised, controlled, open-label, phase 3 trial. Lancet Oncol. 2015 Apr;16(4):375–84. Weber J, Mandala M, Del Vecchio M, Gogas HJ, Arance AM, Cowey CL, Dalle S, Schenker M, Chiarion-Sileni V, Marquez-Rodas I, Grob JJ, Butler MO et al. CheckMate 238 Collaborators. Adjuvant Nivolumab versus Ipilimumab in Resected Stage III or IV Melanoma. N Engl J Med. 2017 Nov 9;377(19):1824–1835. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015 Jul 2;373(1):23–34. Hirano T. Interleukin 6 and its receptor: ten years later. Int Rev Immunol. 1998;16(3–4):249–84. Hunter CA, Jones SA. IL-6 as a keystone cytokine in health and disease. Nat Immunol. 2015 May;16(5):448–57. Kumari N, Dwarakanath BS, Das A, Bhatt AN. Role of interleukin-6 in cancer progression and therapeutic resistance. Tumour Biol. 2016 Sep;37(9):11553–11572.
Ethics Approval
This study was approved by the Ethics Committee of Istituto Nazionale Tumori – IRCCS – Fondazione 'G. Pascale', Naples, Italy, protocol number 17/17 oss.
Abstract Background PD-1 blocking agents, such as nivolumab, have demonstrated clear anti-tumor effects and clinical benefits in a subset of patients with advanced malignancies. Nonetheless, more efforts are needed to identify reliable biomarkers for outcome, to correctly select patients who will benefit from anti-PD-1 treatment. The aim of this study was to investigate the role of peripheral CD8+T cells expressing CD73, involved in the generation of the immune suppressive molecule adenosine, in predicting outcome after nivolumab treatment in advanced melanoma patients. Methods PBMCs from 100 melanoma patients treated with nivolumab were collected at National Cancer Institute “G. Pascale” of Naples. Frequencies of CD8+ lymphocytes phenotypes were assessed by flow cytometry at baseline before nivolumab treatment, along with clinical characteristics and blood count parameters. Healthy controls (n = 20) were also analysed. Percentages of baseline T cells expressing PD-1 and CD73 were correlated with outcome after nivolumab treatment. Results Melanoma patients presented a lower frequency of total circulating CD8+ lymphocytes than control subjects (p = 0.008). Patients with low baseline percentage of circulating CD8+PD-1+CD73+ lymphocytes (< 2.3%) had better survival (22.4 months vs 6.9 months, p = 0.001). Patients (39%) with clinical benefit from nivolumab therapy presented a significantly lower frequency of circulating CD8+PD-1+CD73+ lymphocytes than patients who progressed to nivolumab treatment (p = 0.02). Conclusions Our observations suggest that baseline CD73 expression on circulating CD8+PD-1+ lymphocytes appear a promising biomarker of response to anti-PD-1 treatment in melanoma patients. Further investigations are needed for validation and for clarifying its role as prognostic or predictive marker.
The immune checkpoint inhibitors (ICIs) revolutionized cancer therapeutic landscape and substantially improved the survival of patients (pts) with advanced malignancies, especially in skin cancer pts.1–5 IL-6 is a key inflammatory molecule secreted by M2 macrophages after polarization, mediating the progression of pancreatic and colorectal cancer.6 7 The purpose of this study is to retrospectively investigate the relationships between IL-6 and outcome in skin cancer patients treated with immunotherapy.
Methods
IL-6 levels were analyzed in two independent cohorts, in cohort 1 serum IL-6 were evaluated from 386 consecutive skin cancer pts before start ICIs. We included pts with unresectable/metastatic cutaneous squamous cell carcinoma (cSCC) treated with cemiplimab (n=47); pts with resected stage III/IV melanoma (n=98) treated with anti-PD1; pts with metastatic melanoma treated with anti-PD1 (n=139), combo ipi+nivo (n=65) and ipilimumab alone (n=37). IL-6 was measured by Electrochemiluminescence immunoassays (ECLIA) from Cobas C6000 (Roche). In cohort 2 we conducted a gene profile analysis with Nanostring from PBMCs of 121 metastatic melanoma pts. All pts signed informed consent. Patient's characteristics are listed in table 1. ROC curves were used to determine the best cut off. Survival rates were analyzed using the Kaplan-Meier method. Hazard Ratios (HR) and their 95% confidence intervals (CI) were estimated using a Cox regression model.
Results
Among 507 pts, in cohort 1 lower serum IL-6 was associated with a better Progression Free Survival (PFS) 18.67 months (95% CI 16.6–20.7) versus 10.31 months (95% CI 8.5–12.0), HR = 0.45 (CI 0,3–0,5, p<0.0001); Overall Survival (OS) (27.59 months (95% CI 25.9–29.2) versus 20.12 months (95% CI 17.7–22.4), HR = 0.32 (CI 0,23–0,47, p<0.0001) and Overall Response Rate (ORR) (p<0.001). Similarly, IL-6 and previous therapy are associated with OS and PFS in multivariate analysis (p <0.01). We also confirmed the association between IL-6 and outcomes in all subgroups. In cohort 2 we observed a similar trend in pts with lower IL-6 expression. Moreover, higher IL-6 was associated to MAP3K12, EGFR, SELL,FPR1 genes.
Conclusions
We found that lower levels of both serum and gene expression of IL-6 are associated with better OS, PFS and ORR. Furthermore, IL-6 is associated to higher expression of genes relate to cell cycle, proliferation and metastasis. Further investigations are needed to get additional information.
Acknowledgements
This work was supported by the Italian Ministry of Health Ricerca Corrente funds
References
James Larkin, Vanna Chiarion-Sileni, Rene Gonzalez, Jean Jacques Grob, C Lance Cowey et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015 Jul 2;373(1):23–34. Robert C, Long GV, Brady B, Dutriaux C, Maio M, et al. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med. 2015 Jan 22;372(4):320–30. Weber JS, D'Angelo SP, Minor D, Hodi FS, Gutzmer R. Nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4 treatment (CheckMate 037): a randomised, controlled, open-label, phase 3 trial. Lancet Oncol. 2015 Apr;16(4):375–84. Schenker M, Chiarion-Sileni V, Marquez-Rodas I, Grob JJ, Butler MO, et al. CheckMate 238 Collaborators. Adjuvant Nivolumab versus Ipilimumab in Resected Stage III or IV Melanoma. N Engl J Med. 2017 Nov 9;377(19):1824–1835. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015 Jul 2;373(1):23–34. He Z, Wang J, Zhu C, Xu J, Chen P, Jiang X, et al. Exosome-derived FGD5-AS1 promotes tumor-associated macrophage M2 polarization-mediated pancreatic cancer cell proliferation and metastasis. Cancer Lett 2022;548:215751. doi: 10.1016/j.canlet.2022.215751. Li W, Wu Z, Meng W, Zhang C, Cheng M, Chen Y, et al. Blockade of IL-6 inhibits tumor immune evasion and improves anti-PD-1 immunotherapy. Cytokine. 2022;158:155976. doi: 10.1016/j.cyto.2022.155976.
Ethics Approval
This study was approved by the Ethics Committee of National Cancer Institute—IRCCS—Fondazione ''G. Pascale'', Naples, Italy, protocol number 32/22 oss. All patients provided their written informed consent to participate in this study.
Consent
Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer with neuroendocrine features, and it is associated with elevated mortality. The pathogenesis is associated with presence of clonally integrated Merkel cell polyomavirus (MCPyV) or ultraviolet light (UV) exposure.1 The MCPyV causes up to 80% of MCC tumors in North America and Europe.2–4 Recently immunotherapy is having good results,5 the phase 2 trial JAVELIN Merkel 200 indicated that treatment with Avelumab (PDL1 inhibitor) in patients with metastatic MCC pre-treated have a meaningful long-term survival outcomes respect chemotherapy. Moreover, ORRs were highest in patients with high TMB that were also MCPyV−, PD-L1+ or had a greater CD8+ T cell density at the invasive margin.6 In this study, we investigated the biological signatures in patients with MCPyV or not.
Methods
From April 2011 to June 2018, we collected retrospectively 50 FFPE (Formalin-Fixed Paraffin-Embed) from 37 patients with metastatic MCC and 13 tissues from a secondary metastatic site. All patients have appropriately signed informed consent. We performed an immunohistochemistry assays (IHC) for MCPyV and PDL1. In addition, through the NanoString GeoMx DSP (Digital Spatial Profiling), we analysed 11 patients (6 MCPyV+; 5 MCPyV-) with cutaneous metastasis using a 44-plex antibody cocktail. For each slide we selected three different areas: Intratumoral, extratumoral and tumour border, in each area we selected CD4+ and CD8+ cells in 4 different ROIs (Region of Interest). Statistical analysis was performed via Bonferroni correction, P< 0.05 was considered statistically significant for median stratification.
Results
The DSP analysis showed that the tumour border cells have an overexpression of IDO respect intratumoral area (adj. p<0.01). Instead, extratumoral area of MCPyV- patients have a higher expression of B7-H3 respect MCPyV+ as well as FOXP3 is higher in the tumour border of MCPyV+ patients and EpCAM in the intratumoral area (p<0.05). PDL1 is overexpressed in MCPyV+ CD4+ cells respect CD8+ (p<0.05). The IHC assay shown that viral status does not change in multiple metastases and PDL1 is elevated in the tumour border (p<0.05).
Conclusions
In this retrospective study, our preliminary data shown that tumour edge have an important role in the modulations of immune infiltrate and patients with Merkel cell polyomavirus could have a different pathway of immunosuppression compared to patients with non-virus related etiology. Further investigations are needed to get additional information.
Acknowledgements
The study was supported by the Institutional Project "Ricerca Corrente" of Istituto Nazionale Tumori IRCCS Fondazione "G. Pascale" of Napoli, Italy.
References
Kaae J, Hansen AV, Biggar RJ, et al. Merkel cell carcinoma: incidence, mortality, and risk of other cancers. J Natl Cancer Inst 2010 June 2;102(11):793–801. Feng H, Shuda M, Chang Y, et al. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 2008 February 22;319(5866):1096–100. Garneski KM, Warcola AH, Feng Q, et al. Merkel cell polyomavirus is more frequently present in North American than Australian Merkel cell carcinoma tumors. J Invest Dermatol 2009 January;129(1):246–8. Goh G, Walradt T, Markarov V, et al. Mutational landscape of MCPyV-positive and MCPyV-negative Merkel cell carcinomas with implications for immunotherapy. Oncotarget 2016 January 19;7(3):3403–15. Bichakjian CK, Olencki T, Aasi SZ, et al. Merkel cell carcinoma, version 1.2018, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw 2018 June;16(6):742–774. D'Angelo SP, Bhatia S, Brohl AS, et al. Avelumab in patients with previously treated metastatic Merkel cell carcinoma: long-term data and biomarker analyses from the single-arm phase 2 JAVELIN Merkel 200 trial. J Immunother Cancer 2020 May;8(1):e000674.
Ethics Approval
The study was approved by internal ethics board of the Istituto Nazionale Tumori IRCCS Fondazione "G. Pascale" of Napoli Italy, approval number of registry 33/17 OSS.
Consent
Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
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