Standing Watch: Immune activation and failure in melanoma sentinel lymph nodes

Summary:

An unbiased approach to map the sentinel lymph node landscape reveals progressive immune dysfunction associated with micrometastasis in stage I-III cutaneous melanoma patients. Evidence of tumor-induced lymph node dysfunction may motivate new hypotheses for neoadjuvant therapy with potential to reinvigorate endogenous anti-tumor immunity.

In this issue of Clinical Cancer Research, Yaddanapudi and colleagues map the immune landscape of sentinel lymph nodes (SLN) from Stage I-III cutaneous melanoma patients (1). By combining mass cytometry, flow cytometry, and T cell receptor sequencing on a large cohort of fresh SLN biopsies, this study reveals numerical and phenotypic shifts in SLN immune populations as a function of metastasis that support the hypothesis that the SLN is immunosuppressed. An expanded understanding of the immunology of the SLN may guide therapeutic intervention in the neoadjuvant setting.

The SLN is a harbinger of poor prognosis in melanoma, where the presence of occult metastasis is associated with increased rates of recurrence and reduced overall survival. The SLN is the most proximal to a primary lesion, identified by lymphatic mapping at time of wide excision, and therefore the most likely first site of regional metastasis. Tumor-draining LNs are also critical sites of antigen presentation and anti-tumor adaptive immune activation, and yet how an immunological organ is simultaneously a critical site for tumor antigen and permissive for tumor metastasis has remained incompletely understood (2). Studies to date describe a series of immunological changes in the SLN that associate with metastasis, including a reduction in both the number and activated state of dendritic cell populations and an increase in regulatory T cells (2). These observations support the presumption that the SLN undergoes a progressive failure of immune control that permits tumor seeding and may further impact systemic immune surveillance. A complete understanding, however, of the changes that occur in the SLN and their sequence as a function of primary tumor progression is lacking.

To create an unbiased map of the immune microenvironment in human SLNs, Yaddanapudi and colleagues (1) collected 108 fresh SLN samples from Stage I-III cutaneous melanoma patients (67 Stage I/II and 41 Stage III). A small portion of the most radioactive SLN was processed and cryopreserved, while the remainder was pathologically assessed for metastatic burden. Single cell suspensions from fresh specimens were submitted to a panel of assays including mass cytometry, flow cytometry, and T cell receptor sequencing to determine the functional immune phenotype of melanoma SLNs. Using mass cytometry, the authors identify dendritic cell, NK cell, neutrophil, B, and T cell clusters with 20 sub-clusters within the T cell compartment, though interestingly fail to capture macrophages and monocyte populations. Their analyses reveal tumor-driven T, NK, and innate cell signatures including increased effector memory α T cells, clonal expansion, immune checkpoint molecule expression, and γδ T cells in metastatic and reduced NK cell cytolytic function in (Stage III) SLNs (Figure 1).

Figure 1. Determining the immune landscape of melanoma sentinel lymph nodes.

Fresh SLNs were obtained from cutaneous melanoma patients and simultaneously prepared for histopathological analysis and multimodality immunophenotyping. Using mass cytometry, flow cytometry and T cell receptor sequencing the authors find an association with distinct phenotypic and functional shifts in T, NK, and innate immune signatures that associates with tumor presence in SLNs (no tumor, SLN⁻; tumor, SLN⁺). A highly resolved, and unbiased landscape of the SLN immune microenvironment may identify targets for therapeutic reinvigoration of anti-tumor immunity in the neoadjuvant setting.

Of particular relevance is the expression pattern of immune checkpoint molecules, particularly those currently targeted by approved immunotherapeutics (e.g. PD-1 and CTLA-4). CTLA-4 was found to be highly expressed on γδ T cells whereas regulatory T cells showed low levels of surface expression. The highest levels of PD-1 were found on select CD4⁺ and CD8⁺ T cells, notably effector memory CD8⁺ and CD4⁺ T cells (CD45⁻CCR7⁻) and activated CD4⁺ central memory (CD45RA⁻CCR7⁺). In addition, TIGIT and LAG3 were significantly increased on CD8⁺ effector memory T cells in metastatic SLNs. Recent studies have pointed to the potency of targeting immune checkpoint blockade (ICB) to the SLN in preclinical models (2,3) and these human data support the hypothesis that the LN could be a direct target of these therapies. Neoadjuvant strategies that leave the SLN intact may therefore reinvigorate this depot of anti-tumor immunity to drive robust systemic immune surveillance. Consistent with this hypothesis, the Phase 1b OpACIN (NCT02437279) and Phase 2 OpACIN-neo (NCT02977052) studies that tested neoadjuvant ipilimumab plus nivolumab in patients with macroscopic stage III melanoma, exhibited high pathological response rates and durable tumor control in responders with two years of follow up (4).

Notably, however the preclinical studies testing the contribution of the SLN to ICB response have not interrogated the impact of coincident LN metastasis, and therefore, whether ICB would reinvigorate immunity to the same extent in a metastatic SLN is unclear. Data presented in this study indicates a progressive loss of T cell proliferative potential as a result of micrometastatic seeding, including decreased expression of CD27 and CD28, markers associated with in vivo proliferative potential, and increased proportions of differentiated CD8⁺CD57⁺PD-1⁺ T cells with high expression of the inhibitory receptor KLRG1. The accumulation of this terminally differentiated, and presumed poorly ICB responsive, CD8⁺ T cell population is interestingly correlated with Breslow thickness, a measure of primary tumor growth. Therefore, whether overall tumor burden, or specifically tumor cell presence in the LN, is the main driver of LN CD8⁺ T cell dysfunction remains to be determined. Still, data from the OpACIN trials indicate that responses are feasible even in the presence of palpable LN disease (4) and the information provided here may suggest new avenues for reinvigoration.

Importantly, the authors report increased clonal frequency concordant with effector memory T cell expansion in metastasis positive SLNs and in correlation with primary Breslow thickness. These findings may indicate local antigen presentation that is enhanced in the presence of micrometastatic tumor burden. Activated CD8⁺CD69⁺ cells, however, decrease with metastasis along with a concomitant increase in regulatory T cells, consistent with prior studies. Interestingly, these CD8⁺CD69⁺ T cells might include a population of long-lived, resident T cells, which were recently described to contribute to LN tumor control in mice, and whose LN abundance was associated with outcome in patients (5). Collectively, this shift from activated, protective T cells subsets, to more terminally differentiated T cells is consistent with chronic antigen recognition and progressive immune dysfunction.

While the strength of the study is the large cohort of fresh samples analyzed with paired phenotypic and functional assays, there remain some limitations. Notably, despite the associations made to LN burden and melanoma stage, the authors are not able to directly link the SLN immunophenotype to distant recurrence or overall survival. Given that the current melanoma staging system still fails to perfectly stratify long-term survival in early stage II and III disease (6), how SLN immunobiology actually tracks with distant recurrence will be interesting to explore. Additionally, as with all single cell approaches, biospecimen heterogeneity may introduce sample bias, and the loss of spatial information may disrupt important architectural features that govern collective immune function. Finally, how specific these observations are to melanoma, and in particular its high somatic mutation burden, or if they may replicate in SLNs of other solid tumors is an interesting, open question.

In conclusion, the immunological changes observed by Yaddanapudi and colleagues are consistent with the hypothesis that anti-melanoma immunity is compromised as a function of regional metastasis and it is reasonable to predict that these changes might contribute to distant recurrence. Functional preclinical studies are needed to test the causal relationship between SLN immunosuppression, systemic immune surveillance, and distant metastatic dissemination in order to nominate specific mechanisms for targeted neoadjuvant therapy. This study is therefore a significant step towards identifying the tumor-driven changes in patients that can guide the design of new therapies to reinvigorate anti-melanoma immunity and prevent recurrence.

List of Abbreviations:

CTLA-4

Cytotoxic T-lymphocyte associated protein 4

ICB

Immune checkpoint blockade

KLRG1

Killer cell lectin like receptor G1

LAG-3

Lymphocyte activation gene 3

LN

Lymph node

NK cell

Natural killer cell

PD-1

Programmed cell death protein 1

SLN

Sentinel lymph node

TIGIT

T cell immunoreceptor with Ig and ITIM domains