Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma

Abstract

BackgroundAdoptive T cell therapy (ACT) has shown great promise in melanoma, with over 50 % response rate in patients where autologous tumor-reactive tumor-infiltrating lymphocytes (TIL) can be cultured and expanded. A major limitation of ACT is the inability to generate or expand autologous tumor-reactive TIL in 25–45 % of patients tested. Methods that successfully identify tumors that are not suitable for TIL generation by standard methods would eliminate the costs of fruitless expansion and enable these patients to receive alternate therapy immediately.MethodsMultispectral fluorescent immunohistochemistry with a panel including CD3, CD8, FoxP3, CD163, PD-L1 was used to analyze the tumor microenvironment in 17 patients with melanoma among our 36-patient cohort to predict successful TIL generation. Additionally, we compared tumor fragments and enzymatic digestion of tumor samples for efficiency in generating tumor-reactive TIL.ResultsTumor-reactive TIL were generated from 21/36 (58 %) of melanomas and for 12/13 (92 %) tumors where both enzymatic and fragment methods were compared. TIL generation was successful in 10/13 enzymatic preparations and in 10/13 fragment cultures; combination of both methods resulted in successful generation of autologous tumor-reactive TIL in 12/13 patients. In 17 patients for whom tissue blocks were available, IHC analysis identified that while the presence of CD8+ T cells alone was insufficient to predict successful TIL generation, the CD8+ to FoxP3+ ratio was predictive with a positive-predictive value (PPV) of 91 % and negative-predictive value (NPV) of 86 %. Incorporation of CD163+ macrophage numbers and CD8:PD-L1 ratio did not improve the PPV. However, the NPV could be improved to 100 % by including the ratio of CD8+:PD-L1+ expressing cells.ConclusionThis is the first study to apply 7-color multispectral immunohistochemistry to analyze the immune environment of tumors from patients with melanoma. Assessment of the data using unsupervised hierarchical clustering identified tumors from which we were unable to generate TIL. If substantiated, this immune profile could be applied to select patients for TIL generation. Additionally, this biomarker profile may also indicate a pre-existing immune response, and serve as a predictive biomarker of patients who will respond to checkpoint blockade. We postulate that expanding the spectrum of inhibitory cells and molecules assessed using this technique could guide combination immunotherapy treatments and improve response rates.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-015-0091-z) contains supplementary material, which is available to authorized users.

Multiparametric immune profiling in HPV– oral squamous cell cancer

“…This might be of great impact, as high PD-L1 expression levels can be mediated by external stimuli, including cytokines secreted from immune cells, or intrinsic constitutive expression due to gene amplification (43), utilization of an ectopic promoter by translocation (44), disruption of the 3′ untranslated region of PD-L1 (45), or aberrant signaling pathways (46). Based on our previous data in melanoma (32), we investigated the ratios as well as the spatial relationship between CD8 + T cells and immune suppressors. Adapted from an optimal separation of a test cohort of 34 patients (Figure 6), FoxP3 and PD-L1 expression was analyzed within a 3-lymphocyte-wide distance (30 μm) around CD8 T cells; the numbers of FoxP3 + and PD-L1 + cells were normalized to the total number of CD8 T cells, and this number was equal to the SI.…”

“…Multispectral imaging was performed as previously described in detail in the supplemental section of Feng et al (32) with minor changes, using mAbs directed against PD-L1 (E1L3N, Cell Signaling), CD8 (M239, Spring Bioscience), FoxP3 (236A/E7, Abcam), CD3 (2GV6, Ventana, prediluted, incubation for 45 minutes), CD163 (MRQ26, Ventana), and cytokeratin (AE1/AE3, DAKO, 1:100 dilution, incubation for 45 minutes) as well as DAPI (Invitrogen). TSACy5 (PerkinElmer Inc.), TSA-Cy3 (PerkinElmer Inc.), TSA-FITC (PerkinElmer Inc.), TSA-Alexa594 (Life Technologies), TSA-Biotin-Alexa514 (PerkinElmer Inc., Life Technologies), and TSA-Coumarin (PerkinElmer Inc.) were applied to each antibody.…”

“…In order to get better insights into these processes and their effect on patient survival, which might help in selecting patients with rather adverse prognosis for specific immunotherapies, this study analyzed different components of immune surveillance/escape using multispectral imaging and objective assessment combined with conventional IHC (32,33).…”

Multispectral Imaging of T and B Cells in Murine Spleen and Tumor

“…Much of the work has been done on human tissues as the staining of various epitopes on FFPE tissues has been optimized (4,22). For murine studies, characterization of the immune distribution in the tissue by IHC is lacking because many epitopes, notably CD4 and CD8, are sensitive to cross-linking and are unable to be detected in FFPE samples.…”

“…Our previous work showed that a TSA-based multiplex fluorescent imaging platform can be used to predict the success of culturing tumor-infiltrating lymphocytes from human melanoma samples (22). A simple diagram of this protocol is shown in Supplemental Fig.…”