4. Discussion
In this single institution, retrospective study we identified an association of involvement of deep margins at biopsy with residual melanoma at the time of wide local excision. Ulceration and absence of TILs at biopsy for stage I and II melanoma patients were independently associated with increased risk for disease progression. Notably, the hazard ratio for absence of TILs was 9 using the best Cox-proportional hazards model, suggesting that patients with absent TILs have a 9x increased risk of progression relative to patients with present TILs. That is over half the hazard ratio for ulceration, which is a well-established risk factor incorporated into staging, found by the same model. While the number of absent TIL patients in our study was small, the magnitude of the effect suggests it warrants further study.
The recent Keynote-716 study suggests that some lower stage melanoma patients could benefit from early adjuvant immunotherapy [8]. The identification of absent TILs could be used as a readily available indicator of high-risk disease in stage I-II patients who may be more likely to benefit from early intervention. The caveat is that commonly used immunotherapies rely on lymphocytic infiltration for clinical efficacy [20-22]. Thus, patients with absent TILs may also be resistant to immune checkpoint inhibitors, which in part could explain their worse overall prognosis.
An alternative approach is to consider absent TILs indicative of a “cold” melanoma. Though melanoma is conventionally considered a “hot” tumor, patients with absent TILs may benefit from strategies that convert immunologically “cold” tumors to “hot” [23]. Incorporating agents that boost immune infiltration into the tumor are likely essential for these patients to benefit from immunotherapy. Zakharia et al combined indoximod, an indoleamine 2,3-dioxygenase inhibitor (IDO) inhibitor, with pembrolizumab in patients with advanced melanoma, resulting in an objective response rate of 51% [24]. IDO depletes tryptophan from the extracellular environment, and increased expression of IDO has been shown to decrease T cell infiltration in colorectal cancer [25]. The TLR-7/8 agonist imiquimod has been demonstrated to increase T cell infiltration and activation for squamous cell carcinoma of the skin [26]. Imiquimod has also been used for melanoma-in-situ with positive margins with an estimated 95% resolution of residual melanoma-in-situ [27]. Case series suggest that imiquimod stimulates T cell infiltration in the context of melanoma-in-situ [28], raising the possibility that imiquimod could improve the outcomes of patients with absent TILs on biopsy. Imiquimod is well tolerated with few side effects and warrants consideration as neoadjuvant treatment for early stage melanomas [29]. Treatment with agents like imiquimod to enhance T cell tumor infiltration, either as monotherapy or in combination with immunotherapy, may improve outcomes in stage I and II patients with absent TILs.
Our study has several limitations. A major limitation is the small sample size (68 patients), which is further compounded by missing data from some biopsy reports (Table 2 ). Furthermore, the relatively small sample size prevented us from evaluating associations between lymphocyte status and response to immunotherapy in patients that recurred. Despite this limitation, our results suggest absent lymphocytes and ulceration identify early stage melanoma patients at higher risk for progression and should be confirmed in a larger study. To do this, we are expanding the scope of this retrospective study to include all evaluable patients within a clinical research database available at our institution. This larger, multi-institutional study could elucidate whether absent lymphocyte status predicts tumor resistance to immune checkpoint inhibitors in early stage patients. Like all retrospective studies, our analysis identifies associations, but we cannot infer causal relationships. Additionally, this study only analyzes patients seen at the an academic tertiary care center. Cheraghlou et al demonstrated that treatment for early stage melanoma at hospitals with an academic affiliation or high-volume is associated improved survival compared to those treated at non-academic or low-volume centers [30]. A prospective study treating patients with imiquimod after wide local excision would determine 1) if absent TIL status can be reversed pharmacologically and 2) whether recruitment of TILs improves progression free survival in early stage melanoma patients.
5. Author contributions:KM contributed to conceptualization, study design, data collection, data interpretation, manuscript writing and review. TE, VA, and EM contributed to data collection, data interpretation and manuscript writing. ZG and JHL performed statistical analyses and manuscript review. BD contributed to conceptualization, study design, data interpretation, manuscript writing, and manuscript review. All authors approved the final version of the manuscript for submission.
6. Data availability: The de-identified datasets generated during and/or analysed during the current study are available from the corresponding author (BD) on reasonable request.