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842 Development of a multiplex test for predicting response to combined immunotherapies in patients with metastatic melanoma
  1. Gabriele Madonna1,
  2. Pedro Machado Almeida2,
  3. Mariaelena Capone1,
  4. Vito Vanella1,
  5. Lucia Festino1,
  6. Antonio Sorrentino2,
  7. Marco Cassano2,
  8. Benjamin Pelz2,
  9. Diego Dupouy2 and
  10. Paolo Ascierto1
  1. 1Istituto Nazionale Tumori IRCCS Fondazione “G. Pascale”, Napoli, Italy
  2. 2Lunaphore Technologies, Tolochenaz, Switzerland


Background In recent years, advanced melanoma treatment has improved dramatically thanks to the advent of immunotherapy. Particularly immune checkpoint inhibitors (ICI), in some patients, have demonstrated to improve long-term outcomes associated with limited toxicity. However, only a small population of patients achieve a durable response to therapy, owing to the lack of clinically validated predictive biomarkers (reviewed in1). The availability of improved predictive biomarkers may allow the identification of patients who will most benefit from ICI treatment and those who may be susceptible to immune-related adverse events. This difficulty in obtaining clinically relevant predictive biomarkers underscores the complexity of the immune system and the heterogeneity of the tumor microenvironment. In the present study, we take the first steps towards stratification of advanced melanoma patients who received a combination of ICI. We studied the predictive value of a multi-parameter spatial signature composed of lymphocyte-activation gene-3 (LAG-3), programmed death-ligand 1 (PD-L1) and cluster of differentiation 8 (CD8) in a retrospective cohort of patients with metastatic melanoma treated with a combination of ICI.

Methods In this retrospective study, from the biobank of the Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, we recovered FFPE skin metastasis samples obtained from 10 melanoma patients with AJCC 8th edition stage IV2 subsequently treated with combined ICI, enrolled from September 2016 to November 2017. According to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1),34 patients achieved response to the treatment, while 6 patients were non-responders. A single FFPE tumor tissue of each patient was stained using LabSat® platform (Lunaphore Technologies) performing a Tyramide Signal Amplification multiplex immunohistochemistry staining for PD-L1, CD8, and LAG-3 expression, followed by DAPI counterstaining and whole slide fluorescent scanning. Single cell segmentation, phenotyping and quantification were performed in QuPath (0.3.0). The workflow is depicted in figure 1.

Results Responder patients showed a statistically significant increase in CD8+ single-positive cell frequency compared to non-responders (figure. 2A and 2C). Non-responder patients displayed a statistically significant increase of PD-L1+ single-positive cell frequency, as well as statistically significant increased frequency of double CD8+PD-L1+ positive cells, previously found to be a poor prognostic in multiple cancer types,4,5 and triple positive cells (figure 2B and 2C).

Conclusions We show here preliminary evidence of the predictive value of a spatial biomarker signature in patients who underwent combined ICI therapy for advanced melanoma. To further demonstrate clinical relevance, a more detailed analysis using larger retrospective and prospective cohorts is ongoing.


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Ethics Approval This study was conducted in accordance with the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of the Istituto Nazionale Tumori IRCCS Fondazione "G. Pascale" (Naples, Italy) (protocol code 33/17oss, approved on 10 January 2018).

Abstract 842 Figure 1

Visual representation of the study presented here. A cohort of 10 advanced melanoma patients underwent biopsies of skin metastasis (left panels). The cohort then followed a full course of combined immune-checkpoint inhibitor immunotherapies (upper middle panels). Based on their clinical response to the combined treatment, the cohort was subdivided in Responders (R) and Non-Responders (NR) (note: disease free survival chart for representation purpose only, not based on real data analysis from this our study). In parallel (bottom middle panels), a single FFPE tissue sample slide from tissue collected prior the start of the treatment underwent automated multiplex immunohistochemistry staining for LAG-3, programmed death-ligand 1 (PD-L1) and cluster of differentiation 8 (CD8) on a LabSat® (Lunaphore) instrument. All samples underwent whole slide imaging (WSI). Finally, a retrospective data analysis quantified the number of positive cells for given markers from the WSI to determine relevant biomarkers discriminating R to NR patients. This strategy is now being scaled up with a larger cohort and a larger panel of markers.

Abstract 842 Figure 2

Multiplex staining and quantification on advanced melanoma(A-B) Representative region of interest of Tyramide Signal Amplification multiplex immunohistochemistry staining for lymphocyte-activation gene-3 (LAG-3, magenta), programmed death-ligand 1 (PD-L1, green), cluster of differentiation 8 (CD8, red) and DAPI (blue) on FFPE melanoma skin metastasis samples obtained from patients prior to their first cycle of combined immune checkpoint inhibitors (ICI) treatment. Three patients were classified as Responder (R) (A) and three as Non-Responder (NR) (B) based on their response evaluation criteria. Scale bar: 20 μm. Yellow arrows (A-B) indicates examples of double-positive CD8+PD-L1+ cells, prominently present in NR patients (B-C). (C) Quantification of the number of CD8+ (upper left), PD-L1+ (upper right), CD8+PD-L1+(bottom left) and CD8+LAG-3+PD-L1+ (bottom right) cells from R and NR patients. * p<0.05, *** p<0.001, Unpaired t-test

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