case_images: PMC6735672_01
case_images: PMC6735672_02
case_images: Radiological evaluation timeline. (A) Start anti-PD-1 treatment, (B) first assessment at 9 weeks and (C) last evaluation visit at 45 weeks. 18F-FDG PET, 2-deoxy-2-(fluorine-18)fluoro-D-glucose positron emission tomography; fusion, 18F-FDG PET/CT fusion image; PD-1, programmed death 1; PR, partial response; TL, target lesion.
case_images: esmoopen-2019-000536f02.jpg
case_images: PMC6735672_02_esmoopen-2019-000536f02.jpg
case_images: F2
case_images: At baseline radiological assessment, three target lesions according to Response Evaluation Criteria in Solid Tumours V.1.1 (RECIST 1.1) were observed: a lymph-node located behind the sternal manubrium of 22 mm in shorter perpendicular axis (target lesion (TL) 1), a wide upper mediastinal lesion of 42 mm in the longest diameter (TL2) and a supradiaphragmatic costovertebral lesion of 28 mm in the longest diameter (TL3) (figure 2A). At first radiological assessment at 9 weeks (W9), imaging showed a partial response (PR) according to RECIST V.1.1 (figure 2B) and no pathological 18F-FDG uptake was found. At next assessments, at 18, 27, 36 and 45 weeks, PR was confirmed and 18F-FDG PET/CT continued to be negative (figure 2C).
case_images: PMC6735672_03
case_images: Example of growth of 3D culture after seeding in Matrigel in the timeline of 14 days without treatment (A) and localisation of PD-L1 protein by immunohistochemistry both in spheroids (B, a-c) and in lung biopsy sample (B, d-f). Scale bar: 10 microm. Localisation by immunofluorescence of PD-L1, vimentin and CD45 markers in patient-derived tumour spheroids (C). Images represent different tumour spheroids deriving from the same patient sample: 4',6-diamidino-2-phenylindole-fluorescent nuclear staining (blue), vimentin and CD45 staining (VIM and CD45, red), PD-L1 staining (green). Scale bar: 20 microm. Results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in 3D ex vivo culture from the patient's surgical sample with indicated treatments. Results are shown as analysis of cell proliferation during treatments (D). Dose-response curve of pembrolizumab. Data are the median of three separate experiments, asterisks indicate significance differences (**p<0.05). 3D, three dimensional; CDDP, cisplatin; CTR, untreated control; PD-L1, programmed death1 ligand; VNR, vinorelbine.
case_images: esmoopen-2019-000536f03.jpg
case_images: PMC6735672_04_esmoopen-2019-000536f03.jpg
case_images: F3
case_images: We first verified the presence of PD-L1 by localising the protein by immunohistochemistry both in the spheroids (figure 3B,a-c) and in the lung biopsy sample (figure 3B,d-f). As shown in figure 3C, immunofluorescence detected high levels of expression of both PD-L1 and vimentin in the primary tumour (as documented in the pathology report) as well as in tumour spheroids. We next demonstrated that patient-derived tumour spheroids were made of a mixture of cancer cells as well as of immune infiltrating cells, with both CD45+ cells and PD-L1 cells (figure 3C). In particular, the vitality of the spheroids was reduced by 10.1% and 35.8%, respectively, while the combination of the two drugs resulted in a more effective spheroid growth inhibition (41.1%), suggesting that the combined treatment could be a better treatment option (figure 3D). Similarly, we also tested pembrolizumab and found that it reduced the viability of tumour spheroids in a dose-dependent manner, reaching the maximum of antiproliferative activity at 10 microg/mL dose (figure 3D), which correspond to the therapeutic dose for clinical use of pembrolizumab.
case_images: PMC6735672_04
case_images: Example of growth of 3D culture after seeding in Matrigel in the timeline of 14 days without treatment (A) and localisation of PD-L1 protein by immunohistochemistry both in spheroids (B, a-c) and in lung biopsy sample (B, d-f). Scale bar: 10 microm. Localisation by immunofluorescence of PD-L1, vimentin and CD45 markers in patient-derived tumour spheroids (C). Images represent different tumour spheroids deriving from the same patient sample: 4',6-diamidino-2-phenylindole-fluorescent nuclear staining (blue), vimentin and CD45 staining (VIM and CD45, red), PD-L1 staining (green). Scale bar: 20 microm. Results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in 3D ex vivo culture from the patient's surgical sample with indicated treatments. Results are shown as analysis of cell proliferation during treatments (D). Dose-response curve of pembrolizumab. Data are the median of three separate experiments, asterisks indicate significance differences (**p<0.05). 3D, three dimensional; CDDP, cisplatin; CTR, untreated control; PD-L1, programmed death1 ligand; VNR, vinorelbine.
case_images: esmoopen-2019-000536f03.jpg
case_images: PMC6735672_04_esmoopen-2019-000536f03.jpg
case_images: F3
case_images: We first verified the presence of PD-L1 by localising the protein by immunohistochemistry both in the spheroids (figure 3B,a-c) and in the lung biopsy sample (figure 3B,d-f). As shown in figure 3C, immunofluorescence detected high levels of expression of both PD-L1 and vimentin in the primary tumour (as documented in the pathology report) as well as in tumour spheroids. We next demonstrated that patient-derived tumour spheroids were made of a mixture of cancer cells as well as of immune infiltrating cells, with both CD45+ cells and PD-L1 cells (figure 3C). In particular, the vitality of the spheroids was reduced by 10.1% and 35.8%, respectively, while the combination of the two drugs resulted in a more effective spheroid growth inhibition (41.1%), suggesting that the combined treatment could be a better treatment option (figure 3D). Similarly, we also tested pembrolizumab and found that it reduced the viability of tumour spheroids in a dose-dependent manner, reaching the maximum of antiproliferative activity at 10 microg/mL dose (figure 3D), which correspond to the therapeutic dose for clinical use of pembrolizumab.
case_images: Spectratypes showing BV-TCR subfamilies from PBMCs and TILs. Synoptic diagram of TCR repertoire analysed in peripheral blood and TILs of the patient. BV families were considered. The white box represents a Gaussian chromatogram (normal distribution); the grey box indicates a skewed repertoire (polyclonal response); the empty box represents the absence of amplification. Some families of PBMCs and TILs showed very similar alterations, as shown in chromatograms of 5.3 and 6.1 BV families (on the right), where the same clones seem to be expanded. BV, beta variable; PBMC, peripheral blood mononuclear cells; TCR, T lymphocyte receptor; TIL, tumour-infiltrating lymphocytes.
case_images: esmoopen-2019-000536f04.jpg
case_images: PMC6735672_04_esmoopen-2019-000536f04.jpg
case_images: F4
case_images: TILs, as shown in figure 4 for families 5.3 and 6.1. These results suggest a potential role of PBMCs' TCR alterations as a surrogate for studying the TCR alterations in TILs; moreover, they further confirm the presence of tumour lymphocyte infiltration, which has the same TCR profile of the peripheral lymphocytes. These data suggest that lymphocyte response is specific for any antigen, underlying a pre-primed T cell response.