PD-L1 assessment in pediatric rhabdomyosarcoma: a pilot study

Rhabdomyosarcoma (RMS) is a highly aggressive tumor arising from immature mesenchymal cells committed to skeletal muscle differentiation, it represents the most frequent soft tissue sarcoma in childhood. Although it is generally responsive to the multimodal therapeutic approaches including intensive chemotherapy, the prognosis of RMS depends on several different variables and for some patients the outcome remains dismal [1].

Pediatric RMS has two major histological subtypes, each with distinct clinical, molecular, and genetic features: the embryonal RMS (ERMS) are more frequent (~ 80% of cases) with a higher incidence in younger children; and the alveolar RMS (ARMS), less frequent (~ 20% of cases) but more aggressive and often resistant to conventional chemo- and radiotherapy, resulting in a 5-year survival rate of only 30% [2‐5]. Specifically, patients with alveolar histology continue to have less than optimal outcome, and most patients with distant metastasis or relapsing disease do not achieve long term cure [6]. Furthermore, long-term survivors may endure from important late functional sequelae related to the burden of multimodal therapies they received. Therefore, the identification and development of more efficient and less toxic therapeutic approaches is absolutely needed [7].

PD-1, a type I transmembrane glycoprotein cell surface receptor expressed on T- and pro-B cells, is an immunoreceptor belonging to the CD28/CTLA-4 family of T-cell regulators and, functioning as an immune checkpoint, plays a critical role in downregulating the immune system by preventing the activation of T-cells. PD-1 binds to two ligands, PD-L1 and PD-L2, which block PD1 receptor e induce PD-1 signaling and T-cell ‘exhaustion’. Recently, targeting the PD-1/PD-L1 immune checkpoint pathway has proved to improve adults patients’ survival, but with less toxicity than conventional treatments, possibly stimulating the anti-tumor immunity by activating the patients’ own immune system [7]. Encouraging clinical benefits of PD-1/PD-L1 checkpoint blockade have been demonstrated in over 15 different malignancies, among which melanoma [8‐10], lung cancer [11, 12], genitourinary tract cancer [13], Hodgkin lymphoma [13] and sarcoma [14] confirming that host immune responses are essential in most neoplasms [15]. As the PD-1 pathway may be a key mechanism of immune escape in a subgroup of patients in several malignancies, PD-L1 expression in tumor or inflammatory cells is a candidate biomarker [12]. However, the only limitation is that PD-L1 status is not effective in identifying the fraction of PD-L1 negative patients that may also benefit from immune therapy [7, 16].

For pediatric malignancies, only a few anti-PD-1 and anti-PD-L1 clinical trials are ongoing and little is known regarding the prognostic and predictive implications of PD-L1 in childhood tumors, in particular RMS. Moreover, to our knowledge, no responses have been reported to anti-PD1 or anti-PD-L1 as a single drug in RMS. Thereby, in an attempt to further describe the immune environment of RMS, we evaluated PD-L1 expression, in a cohort of 25 RMS specimens utilizing two anti-PD-L1 antibodies by immunohistochemistry (IHC) approach on Formalin-Fixed Paraffin Embedded (FFPE) RMS tissues and cytoblocks from RMS cell lines. Our observations were further confirmed by flow cytometry analysis in RMS cell lines, both commercial and primary cultures derived from surgical RMS specimens.

Successful results obtained with pembrolizumab and nivolumab in melanoma, NSCLC, sarcoma and other malignancies [10‐12, 17, 18], have been recently reported, bringing forward immune checkpoint inhibitors as potential treatment options. Since there has been limited research to investigate the clinical and prognostic significance of the PD-1/PD-L1 axis in pediatric malignancies, in the present study we assess the presence of PD-L1 expression in pediatric RMS primary tumors and corresponding cell lines. Our results identify PD-L1 expression in 60% of RMS analyzed, detecting mild/moderate staining uniquely in the immune cells surrounding the tumor burden and/or in those infiltrating the tumor, thus never observing expression in the neoplastic cells.

Current studies have pointed out that high expression of PD-L1 in tumor cells is associated with poor prognosis in NSCLC [11, 12, 19], ovarian cancer [20] and kidney cancer [21], melanoma [22], renal cancer [21], Hodgkin lymphoma [16] and bone and soft tissue sarcoma [15, 23, 24]. Our study demonstrated, by using two different anti-PD-L1 clones that PD-L1 expression is confined in immune cells infiltrating and/or surrounding the tumor burden, but not in RMS tumor cells. To further confirm our results, we assessed PD-L1 expression of RMS cell lines by two different techniques (flow cytometry and IHC on cytoblocks) and using two different clones of anti-PD-L1 antibodies: we were able to detect a very low percentage of tumor cells by flow cytometry, due to the high sensitivity of this technique, that accordingly was hardly detectable with a conventional IHC approach on FFPE cytoblocks. This data confirms the almost completely absence of PD-L1 expression in RMS tumor cells, even when different techniques and antibody clones were utilized. Low expression of PD-L1 in RMS was also reported by Torabi et al. detecting positivity in 3/96 cases in a TMA [24]. Our data are in contrast with those reported by Kim et al. showing an expression of PD-L1 in tumor cells in 37% (12/32) of specimens [15]. However, discordant observations in expression of PD-L1 could be explained by use of different anti-PD-L1 antibodies, staining procedures, and antigen retrieval techniques.

Interestingly, an important subdivision of anticancer immunity in humans into three main phenotypes is represented by Chen & Mellman: immune-desert, immune-excluded and immune-inflamed. Accordingly to this classification, our observations enabled us to subdivide our cohort in 4 ‘immune-inflamed’ RMS, displaying expression of PDL1 in immune cells surrounding and within the tumor burden, which may likely respond to anti-PD-L1/PD-1 therapy, and 5 ‘immune-excluded’ RMS with PD-L1 staining present in immune cells that do not penetrate the parenchyma of the tumor but rather are retained in the surrounding stroma, which are expected to rarely respond to PD-L1/PD-1 agents [25]. At last, 7 specimen were revealed to be ‘immune-desert’ and characterized by very few T-cells in either the parenchyma or the stroma of the tumor burden, therefore not responsive to PD-L1/PD-1 agents [25].

Dynamic changes of PD-L1 protein expression were observed in 4 RMS tumors evaluated at diagnoses/first progression of disease and at a (second) tumor progression post-treatment, suggesting that damages induced by chemotherapy treatments in tumor cells and stroma may foster an inflammatory microenvironment and recruitment of PD-L1 expressing immune cells, creating an immune contexture possibly druggable by immune checkpoint inhibitors. Indeed randomized clinical trial using immunotherapy in second line after chemotherapeutic treatments in NSCLC indicated successful response rate [12, 26]. These results also reinforce the concept to perform biopsies, when possible, prior of initiating an immune checkpoint treatment to confirm the immune contexture status at that particular time, as it is easily influenced and prone to changes [even if the biomarker validity of PD-L1 positivity is highly debatable].

In view of our results, the lack of expression by the neoplastic cells discourages us to consider RMS an immunogenic tumor possibly explaining the lack of literature of immune checkpoint inhibitors in pediatric RMS. However, the distinct PD-L1 expression pattern observed in our cohort, could be critical in discriminating ‘immune-inflamed’ from ‘immune-excluded’ specimen, making a significant difference in discriminating those patients that may benefit from PD-1/PD-L1 therapy.

To the date, clinical trials with immune checkpoint inhibitors in pediatric malignancies are few and with mainly unsatisfactory results, thus an effort to characterize the immune contexture is needed. In this study, we demonstrate by different techniques and in multiples setting the low expression of PDL1 by RMS, and we observed a possible complementary role of chemotherapy as igniter of ‘inflamed tumors’. Taken altogether these data may suggest the possibility of a combination with conventional chemotherapy and PD-L1 checkpoint blockade.