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Histology of microdissected lymphocytes in primary malignant melanoma.Primary malignant melanoma with clustered TIL and dissected clusters of lymphocytes.
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It is established that primary malignant melanomas (pMM) can be infiltrated by T-cell populations with predominantly one T-cell clone. As pMM generally express multiple tumor-associated antigens (TAA), here we used laser-capture microdissection (LCM) to isolate different tumor-infiltrating lymphocyte (TIL) clusters in order to determine whether pMM...
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... 21 malignant melanoma specimens from 21 patients with a prominent lymphocytic infiltrate (Table 3), one 10-mm-thick tissue section was cut from the paraffin-embedded tissue block and stained using hematoxylin. Two lymphocytic infiltrates (approximately 500-2000 cells each) from various regions of the section, which were not adjacent to each other, were microdissected (Figure 1) with a PixCell II Laser Capture Microdissection System (Arcturus Engineering Inc., Mountain View, CA). The captured cells were immediately transferred to proteinase K-enriched digestion buffer (0.1 M Tris-HCl, 0.5 mM EDTA, 0.5% Tween 20). ...
Citations
... Yazdi et al. showed that heterogenous T-cell clones could infiltrate primary melanomas. 2 TILs include CD8+ T, CD4+ T, B cells, and NK cells, with CD8+ T cells being the most common subtype in melanoma and associated with a better prognosis. In contrast, other immune cells, including M2 macrophages, T-regulatory cells (Treg), and myeloid-derived suppressor cells (MDSC), act immunosuppressive, leading to tumour promotion. ...
Background: Recent advances in digital pathology have enabled accurate and standardised enumeration of tumour-infiltrating lymphocytes (TILs). Here, we aim to evaluate TILs as a percentage electronic TIL score (eTILs) and investigate its prognostic and predictive relevance in cutaneous melanoma. Methods: We included stage I to IV cutaneous melanoma patients and used hematoxylin-eosin-stained slides for TIL analysis. We assessed eTILs as a continuous and categorical variable using the published cut-off of 16.6% and applied Cox regression models to evaluate associations of eTILs with relapse-free, distant metastasis-free, and overall survival. We compared eTILs of the primaries with matched metastasis. Moreover, we assessed the predictive relevance of eTILs in therapy-naïve metastases according to the first-line therapy. Findings: We analysed 321 primary cutaneous melanomas and 191 metastatic samples. In simple Cox regression, tumour thickness (p
... γδT cells have been identified within tumor-infiltrating lymphocytes (TIL) in multiple cancers 19 but their clinical relevance remained mostly unknown. Regarding melanoma, it has been shown that γδT cells infiltrate primary tumors, 20,21 but little information regarding their features and clinical significance has been reported. Lower proportions of circulating γδT cells especially Vδ2+ cells and higher proportions of Vδ1+ cells have been reported in melanoma patients. ...
... Within γδT cells, higher proportions of tumor-infiltrating δ2+ cells showed a trend toward longer PFS and OS. This is in line with other studies describing that γδT cells including δ2+ infiltrate primary melanoma tumor, representing 15-25% of TIL, 20,30 and their proportion correlated negatively with disease stage 21 and positively with progression-free and overall survival. 26,31 Furthermore TCRγ-/-mice lacking γδT cells are highly sensitive to tumor development upon exposure to chemical carcinogens or tumor inoculation. ...
γδT cells hold a pivotal role in tumor immunosurveillance through their prompt activation and cytokine secretion, their ability to kill tumor cells in an Human Leukocyte Antigen (HLA)-unrestricted manner, and their combination of features of both innate and adaptive immunity. These unique properties and functional plasticity render them very attractive both as targets and vectors for cancer immunotherapy. Yet, these potent and fascinating antitumor effectors have not been extensively explored in melanoma. We provided here a detailed investigation of the phenotypic and functional properties of circulating and tumor-infiltrating γδT cells in melanoma patients, and their impact on clinical evolution. High proportions of circulating- and tumor-infiltrating γδT and δ2+ subset were associated with better clinical outcome. We reported however that circulating and tumor-infiltrating γδT cells from melanoma patients displayed an altered expression of NCR, KIR, and immune checkpoints, and identified NKp44, PD1, 41BB/41BBL, TIM3, and LAG3 as crucial checkpoints allowing immune escape and tumor progression. Notably, melanoma drastically impaired the ability of γδT cells to exhibit activation molecules, secrete cytokines, and display cytotoxicity toward melanoma in response to stimulation with phosphoantigens. It drove them toward regulatory and Th17 profiles associated with poor clinical outcomes. Our study highlights that melanoma hijacked γδT cells to escape from immune control, and revealed that circulating and tumor-infiltrating γδT cell features are promising potential biomarkers of clinical evolution. Such understanding of the physiopathology of γδT cells may help designing new therapeutic approaches exploiting the antitumor potential of γδT cells while counteracting their skewing by tumors to improve patient outcomes.
... Bereits vor vielen Jahren wurden die immunologischen Eigenscha en des malignen Melanoms untersucht. Dabei wurde nachgewiesen, dass es verschiedene tumorassoziierte Antigene exprimiert [9] und gleichzeitig die spezi sche T-Zell-Antwort unterdrückt. Zu einer Spontanremission kommt es daher selten. ...
Durch zielgerichtete Behandlungsoptionen und Immuncheckpointinhibitoren wurde die Prognose von Patienten mit metastasiertem Melanom deutlich verbessert. Dennoch bedarf es weiterer Forschungsbestrebungen. Zurzeit stehen zum Beispiel verschiedene Kombinationen von Checkpointinhibitoren untereinander und mit anderen Substanzen im Fokus der Forscher.
... Bereits vor vielen Jahren wurden die immunologischen Eigenscha en des malignen Melanoms untersucht. Dabei wurde nachgewiesen, dass es verschiedene tumorassoziierte Antigene exprimiert [9] und gleichzeitig die spezi sche T-Zell-Antwort unterdrückt. Zu einer Spontanremission kommt es daher selten. ...
Durch zielgerichtete Behandlungsoptionen und Immuncheckpointinhibitoren wurde die Prognose von Patienten mit metastasiertem Melanom deutlich verbessert. Dennoch bedarf es weiterer Forschungsbestrebungen. Zurzeit stehen zum Beispiel verschiedene Kombinationen von Checkpointinhibitoren untereinander und mit anderen Substanzen im Fokus der Forscher.
... As cells with the T RM phenotype are known to remain in tissues without equilibration in parabiotic mice, it may help explain why the TCR sequences at individual metastases can differ significantly. Differences in TCRβ usages in individual melanoma metastases were also previously noted before the biology of T RM cells was known (24, 25). ...
Heterogeneity of tumor cells and their microenvironment can affect outcome in cancer. Blockade of immune checkpoints (ICPs) expressed only on a subset of immune cells leads to durable responses in advanced melanoma. Tissue-resident memory T (TRM) cells have recently emerged as a distinct subset of memory T cells in nonlymphoid tissues. Here, we show that functional properties and expression of ICPs within tumor-infiltrating lymphocytes (TILs) differ from those of blood T cells. TILs secrete less IL-2, IFN-γ, and TNF-α compared with circulating counterparts, and expression of VEGF correlated with reduced TIL infiltration. Within tumors, ICPs are particularly enriched within T cells with phenotype and genomic features of TRM cells and the CD16(+) subset of myeloid cells. Concurrent T cell receptor (TCR) and tumor exome sequencing of individual metastases in the same patient revealed that interlesional diversity of TCRs exceeded differences in mutation/neoantigen load in tumor cells. These findings suggest that the TRM subset of TILs may be the major target of ICP blockade and illustrate interlesional diversity of tissue-resident TCRs within individual metastases, which did not equilibrate between metastases and may differentially affect the outcome of immune therapy at each site.
... The role of gd T-cells in melanoma is poorly understood thus far. These cells do infiltrate primary melanomas, suggesting that they may be exerting antitumour activity [27]. Only a few studies have reported alterations in frequencies and functional impairment of peripheral gd T-cells in melanoma patients compared to healthy controls but there is as yet no consensus on this [28e30]. ...
... Two important observations have shaped melanoma immunotherapy: (i) On principle, melanomas may regress with and rarely also without immunotherapy, and may sometimes even completely resolve (Figure 1a, b). This regression is usually associated with an oligoclonal T-cell infiltrate [17]. On immunotherapy, melanomas and melanoma metastases constantly regress, either partially or completely. ...
Forty years of research have brought about the development of antibodies that induce effective antitumor immune responses through sustained activation of the immune system. These "immune checkpoint inhibitors" are directed against immune inhibitory molecules, such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1) or programmed death ligand 1 (PD-L1). Disruption of the PD-1/PD-L1 interaction improves the intermediate-term prognosis even in patients with advanced stage IV melanoma. One and a halfyears after treatment initiation, 30-60 % of these patients are still alive. While cancer immunotherapies usually do not eradicate metastases completely, they do cause a regression by 20-80 %. It is well established that the immune system is able to kill tumor cells, and this has also been demonstrated for immunotherapies. Preclinical data, however, has shown that anti-cancer immunity is not limited to killing cancer cells. Thus, through interferon gamma and tumor necrosis factor, the immune system is able to induce stable tumor growth arrest, referred to as senescence. Ensuring patient survival by long-term stabilization of metastatic growth will therefore become a central goal of antitumor immunotherapies. This therapeutic approach is effective in melanoma and non-small-cell lung cancer. Once immunotherapies also have an indication for common cancer types, drug prices will have to drop considerably in order to be able to keep them available to those dependent on such therapies.
... Zwei wichtige Beobachtungen haben die Melanom-Immuntherapie geprägt: (i) Grundsätzlich können Melanome mit und selten auch ohne Immuntherapie in eine Regression übergehen und sich manchmal sogar zurückbilden (Abbildung 1a, b). Diese Regression ist in der Regel mit einem oligoklonalen T-Zell-Infiltrat assoziiert [17]. Unter einer Immuntherapie bilden sich Melanome und Melanommetastasen immer wieder teilweise oder vollständig zurück. ...
Zusammenfassung
Die Erkenntnisse aus 40 Jahren Forschung erlauben es, durch Antikörper‐vermittelte Aktivierung des Immunsystems eine therapeutisch wirksame Antitumorantwort zu induzieren. Die „Immun‐Checkpoint‐Inhibitoren“ sind gegen immuninhibitorische Moleküle wie cytotoxic T lymphocyte antigen 4 (CTLA4), programmed‐death‐1 (PD‐1) oder programmed‐death‐ligand‐1 (PD‐L1) gerichtet. Die Unterbrechung der PD‐1/PD‐L1‐Interaktion verbessert mittelfristig auch die Prognose bei Patienten mit Melanomen im Stadium IV. So sind unter Therapie mit Anti‐PD‐1‐Antikörpern 30–60 % dieser Patienten nach eineinhalb Jahren am Leben. Antitumorale Immuntherapien verursachen nur selten eine vollständige Zerstörung der Metastasen, sondern eine Regression um 20–80 %. Fest etabliert ist, dass das Immunsystem Tumorzellen abtöten kann; dies wurde auch für Immuntherapien belegt. Präklinische Daten zeigten jedoch, dass Immunantworten Tumoren nicht nur töten können. So kann das Immunsystem über die Zytokine Interferon‐γ und Tumornekrosefaktor in Tumoren einen stabilen Wachstumsarrest hervorrufen, der Seneszenz genannt wird. Ein Ziel antitumoraler Immuntherapien wird also darin liegen, das Überleben der Patienten durch eine langfristige Stabilisierung der Metastasen zu sichern. Dies gilt für das Melanom und das nichtkleinzellige Lungenkarzinom. Sobald Immuntherapien auch bei häufigen Tumoren indiziert sein werden, müssen die Medikamentenpreise deutlich fallen, um sie weiterhin jenen zur Verfügung stellen zu können, die die Therapien benötigen.
... A wide variety of TCR rearrangement patterns in TIL-iPSC clones suggests heterogeneity of T cell clones in melanoma tumors. This result is in support of the study from Yazdi et al., who used laser-capture microdissection to isolate different TIL clusters from melanoma and demonstrated that many different T cell clones with different rearrangements were detected within the same individual [42]. ...
Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate
in vitro
and
in vivo
. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy.
... 24,25 Our results confirms previous findings from Yazdi et al., who used laser-capture micro-dissection to isolate different TIL clusters and demonstrated that many T-cell clones with different TCR rearrangements may be detected within one primary malignant melanoma. 26 The significant increase in aggregate clonality upon the administration of BRAFi suggests that other factors are involved in response to treatment. The kinetics of such an increase in clonality (after 10-14 d) is not surprising, as Buchholz et al. have shown that a spectrum of T-cell expansion kinetics exists, ranging from slow-dividing, long-lived T cells to fast-proliferating, short-lived cell subsets in which a single T lymphocyte can lead to 70,000 descendants in infection models. ...
There have been significant advances with regard to BRAF-targeted therapies against metastatic melanoma. However, the majority of patients receiving BRAF inhibitors (BRAFi) manifest disease progression within a year. We have recently shown that melanoma patients treated with BRAFi exhibit an increase in melanoma-associated antigens and in CD8+ tumor-infiltrating lymphocytes in response to therapy. To characterize such a T-cell infiltrate, we analyzed the complementarity-determining region 3 (CDR3) of rearranged T-cell receptor (TCR) β chain-coding genes in tumor biopsies obtained before the initiation of BRAFi and 10-14 d later. We observed an increase in the clonality of tumor-infiltrating lymphocytes in 7 of 8 patients receiving BRAFi, with a statistically significant 21% aggregate increase in clonality. Over 80% of individual T-cell clones detected after initiation of BRAFi treatment were new clones. Interestingly, the comparison of tumor infiltrates with clinical responses revealed that patients who had a high proportion of pre-existing dominant clones after the administration of BRAFi responded better to therapy than patients who had a low proportion of such pre-existing dominant clones following BRAFi. These data suggest that although the inhibition of BRAF in melanoma patients results in tumor infiltration by new lymphocytes, the response to treatment appears to be related to the presence of a pre-existing population of tumor-infiltrating T-cell clones.
