Expression of PD-L1 in malignant soft tissue neoplasm and their correlation with clinicopathological parameters

INTRODUCTION

Soft tissue tumors represent complex and heterogeneous mesenchymal neoplasms with a varied range of differentiation. Histopathological classification based on morphological findings often demonstrates the differentiation of specific cell lines. Soft tissue sarcomas account for less than 1% of the overall cancer burden, in contrast to carcinomas and other malignant neoplasms.^[1] Immune checkpoint inhibitors have shown great promise in the management of a number of malignancies, including melanoma, non-small cell lung carcinoma, renal cell carcinoma, and soft tissue sarcoma. These agents target the anti-programmed death-1 (PD-1) receptor and the anti-programmed death ligand-1 (PD-L1).^[2-4] While PD-L1-targeted treatments have acquired universal recognition for the management of non-small cell lung carcinoma and melanoma, their utility in soft tissue sarcoma is not well established.^[5-8]

Advance sarcoma management is challenging, and anti-immune checkpoint inhibitors have been explored as promising therapeutic strategies. The expression of PD-1 or PD-L1 is typically necessary for PD-L1-targeted treatments to be effective. The expression of PDL-1 in various histological types of sarcomas has been examined in a few studies, although the results are variable. The presence of PD-L1 expression may also have important prognostic consequences.^[9-12] There are numerous studies on PD-L1 expression in malignant epithelial neoplasms, but less data are available on its utility in soft tissue sarcomas.

MATERIALS AND METHODS

This was a prospective study done for the duration of 1 year (November 2019–October 2020) in the Department of Pa-thology, King George’s Medical University, Lucknow, in collaboration with the Department of Surgical Oncology, King George’s Medical University, Lucknow, after getting approval from the Institutional Ethical Committee. All the biopsyand immunohistochemistry-proven cases of malignant soft tissue neoplasms were included in the study. The cases with inadequate biopsy tissue for the application of immunohistochemistry were excluded from the study. Patients who did not provide consent were also excluded from the study population.

The final study included 50 cases of malignant soft tissue neoplasms. For each case, clinical details were recorded. PD-L1 and Ki-67 immunohistochemistry were applied and assessed using standard protocols in each case, in addition to histopathological examination of hematoxylin– eosin (H-E)-stained sections. All the cases of malignant soft tissue neoplasms were morphologically graded as per the French Federation Nationale des Centres de Lutte Contre le Cancer (FNCLCC) grading system. Immunohistochemical staining for PD-L1 and Ki-67 was done on representative sections using the antibody clones E1L3N (Rabbit monoclonal, Ventana, Tucson, Arizona, United States) and DAKO (DSS Imagetech), respectively.

Normal human placental tissue was used as a positive control for PD-L1 and a normal lymph node for the Ki-67 antibody as per the manufacturer’s instructions. The tumor cell percentage with complete or incomplete membrane staining was noted. Sarcomas were categorized as positive (>1% of tumor cells with membrane staining) or negative (≤1% of tumor cells with membrane staining).^[13,14] Ki-67 proliferation index was calculated, and grading was done as grade I (0–9% tumor cells with nuclear positivity), grade II (10–29% tumor cells with nuclear positivity), and grade III (2:30% tumor cells with nuclear positivity).^[15] In each case, PDL1 expression was accessed by two histopathologists with good experience in regular soft tissue reporting. To reduce the subjectivity both the histopathologists were blinded to clinical details and findings of each other results. The pathologists evaluated and discussed cases where there was a disparity until an agreement was achieved.

RESULTS

Association of PD-L1 Immunohistochemical Expression with Histological Grading, Histopathological Diagnosis, and Prognosis

The majority (56.25%) (9/16) of cases with positive PDL1 expression fall into FNCLCC grade II, followed by 25% (4/16) cases in grade III and 18.75% (3/16) cases in grade I. On comparing statistically, FNCLCC grade did not show a significant association with expression of PD-L1 (p = 0.867) (Table 2).

Table 2: Association of PD-L1 immunohistochemical expression with histopathological grading

Histopathological grading	PD-L1 ≤ 1% (n = 34)		PD-L1 >1% (n = 16)
	Number	Percentage (%)	Number	Percentage (%)
Grade I (n = 9)	6	17.65	3	18.75
Grade II (n = 26)	17	50.0	9	56.25
Grade III (n = 15)	11	32.35	4	25.0

Abbreviation: PD-L1, programmed death ligand-1. Chi-square = 0.285 (df = 2); p = 0.867.

The positive expression of PD-L1 was shown in 75% (3/4) cases of undifferentiated pleomorphic sarcoma, followed by 66.67% (2/3) cases of rhabdomyosarcoma, 60.0% (3/5) cases of liposarcoma, 60.0% (3/5) cases of leiomyosarcoma, 16.67% (3/18) cases of synovial sarcoma, 16.67% (1/6) cases of dermatofibrosarcoma protruberens, and 11.11% (1/9) cases of Ewing’s sarcoma. Out of the total 50 cases, 32% (16/50) were positive for PD-L1 expression, and the proportion of expression of PD-L1 was statistically significant in the different histological subtypes of soft tissue sarcomas (p = 0.042) (Table 3) (Figures 1 and 2).

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Figure 1:

Photomicrograph of histopathology section and expression of PD-L1 immunohistochemistry in soft tissue sarcomas. (A) Synovial sarcoma; (B) positive expression of PD-L1 in tumor cells of synovial sarcoma (PD-L1 x400); (C) dermatofibrosarcoma protuberans; (D) positive expression of PD-L1 in tumor cells of dermatofibrosarcoma protuberans (PD-L1 x400); (E) Ewing’s sarcoma; (F) positive expression of PD-L1 in tumor cells of Ewing’s sarcoma (PD-L1 x400). PD-L1, programmed death ligand-1.

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Figure 2:

Photomicrograph of histopathology section and expression of PD-L1 immunohistochemistry in soft tissue sarcomas. (A) Rhabdomyosarcoma; (B) positive expression of PD-L1 in tumor cells of rhabdomyosarcoma (PD-L1 x400); (C) undifferentiated pleomorphic sarcoma; (D) positive expression of PD-L1 in tumor cells of undifferentiated pleomorphic sarcoma (PD-L1 x400); (E) leiomyosarcoma; (F) positive expression of PD-L1 in tumor cells of leiomyosarcoma (PD-L1 x400). PD-L1, programmed death ligand-1.

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Table 3: Association of PD-L1immunohistochemical expression with histopathological diagnosis

Histopathological diagnosis	PD-L1 ≤ 1 (n = 34)		PD-L1 > 1 (n = 16)
	Number	Percentage (%)	Number	Percentage (%)
Synovial sarcoma (n = 18)	15	83.33	3	16.67
Dermatofibrosarcoma protuberans (n = 6)	5	83.33	1	16.67
Ewing’s sarcoma (n = 9)	8	83.89	1	11.11
Leiomyosarcoma (n = 5)	2	40.0	3	60.0
Liposarcoma (n = 5)	2	40.0	3	60.0
Rhabdomyosarcoma (n = 3)	1	33.33	2	66.67
Undifferentiated pleomorphic sarcoma (n = 4)	1	25.0	3	75.0

Abbreviation: PD-L1, programmed death ligand-1. Chi-square = 13.057 (df = 6); p = 0.042,

Fifty percent (8/16) of patients with positive PD-L1 expression had died, and 31.25% (5/16) patients were alive and doing well during a follow-up period of 1 year; 18.75% (3/16) of patients with positive PD-L1 expression were lost to follow-up. A significant association was found for PDL1 expression with the poorer outcome of follow-up (p = 0.024) (Table 4).

Table 4: Association of PD-L1 with outcome of follow-up

Follow-up outcome	PD-L1 ≤ 1 (n = 34)		PD-L1 > 1 (n = 16)
	Number	Percentage (%)	Number	Percentage (%)
Died (n = 13)	5	14.70	8	50.0
Lost to follow-up (n = 18)	15	44.12	3	18.75
Alive (n = 19)	14	41.18	5	31.25

Abbreviation: PD-L1, programmed death ligand-1. Chi-square = 7.440 (df = 2); p = 0.024.

DISCUSSION

In the tumor microenvironment, persistent antigen exposure induces the PD-1 receptor, a particular class of inhibitory signal. Tumor cells are able to evade the host immune system due to the interaction between PD-L1 in tumor cells and PD-1 in T lymphocytes.^[16] PD-1 and PD-L1 are linked to immune checkpoints that favor tumor escape from immune surveillance. Numerous innovative treatments have been designed that target either PD-1 or PD-L1 in light of a growing understanding of these immune checkpoints. Immunotherapies based on checkpoint inhibition are currently used to treat renal cell carcinoma, non-small cell lung cancer, and melanoma.^[17] Immunohistochemical expression of prognostic markers, such as PD-L1 and PD-1, is typically required for the start of therapy with these drugs. The proliferation index (Ki-67) is a nuclear antigen that is used as an immunohistochemical marker to assess the growth percentage in a given cell population, and this protein is seen in all dividing cells.^[15,18,19] The Ki-67 proliferation index was used as a prognostic marker that had been investigated in a number of studies for its potential role in breast cancer, soft tissues arcoma, lung cancer, prostate cancer, and cervix carcinoma.^[20,21]

Among the study population, 68% (34/50) of patients were male and 32% (16/50) were female. A few prior studies have also found a male majority in their studies.^[11,22]

The current study revealed a significant link between positive PD-L1 expression and malignant mesenchymal neoplasms with a more pleomorphic phenotype than other sarcoma subtypes (p = 0.042). The most common soft tissue neoplasm with PD-L1 expression was undifferentiated pleomorphic sarcomas, followed by rhabdomyosarcomas, liposarcomas, leiomyosarcomas, synovial sarcomas, DFSP, and Ewing’s sarcoma. The results of our study are in concordance with previous studies.^[23-25] The results of the present study and the literature review could not identify the cause of the variance in PD-L1 expression seen with distinct subtypes of sarcoma. This variation in PD-L1 expression demonstrates the necessity of immunohistochemistry testing to determine whether PD-L1 is present or not before checkpoint inhibitors are used as a treatment.

In our study, 32% (16/50) of the sarcoma cases were PD-L1 positive. The results of the present research are comparable to those from earlier studies by Kim et al and Paydas et al.^[11,24]

A few previous studies showed relatively reduced expression of PD-L1 in soft tissue sarcoma cases^[13,14,23] (Table 5). Thereported percentages of sarcomas expressing PD-L1 may differ due to sampling size, geographic distribution, the antibodies used, or the cutoff values for positive expression, among possible multifactorial causes.

The correlation between the Ki-67 proliferation index and FNLCC histological grade was statistically significant (p = 0.047) in the present study. The findings of our results were in concordance with previous studies, which also demonstrated the link between high Ki-67 and the histological grade of sarcoma.^[26-28]

In the present study, the association between PD-L1 expression and the Ki-67 proliferation index was found to be significant (p = 0.013); 62.50% (10/16) of patients with positive PD-L1 expression had a high proliferation index. Orth et al also observed a significant correlation between PDL1 expression and the tumor cell proliferation index.^[14]

PD-L1-positive cases showed a statistically significant (p = 0.024) association with poor prognosis in the present study. Que et al observed in their study that PD-L1 expression was linked with short overall survival (p = 0.001).^[22] The results of Kim et al’s and Bertucci et al’s studies are also in concordance with the findings observed in the present study.^[11,12]

PD-L1 treatment is known to have a major impact on clinical outcomes for patients with non-small cell lung carcinoma and melanoma, but we lack data from this study to establish what effect it may have on the clinical course of the patients. Based on these findings, PD-L1 therapy for positive expressors may improve patient survival.

The small sample size, an unequal distribution of histologic subtypes of sarcomas, and a brief follow-up period are the main limitations of our study. It is therefore recommended to conduct studies with larger sample sizes and longer follow-up durations.

CONCLUSIONS

Our study revealed a significant correlation between malignant soft tissue tumor positivity for PD-L1 expression and pleomorphic morphology, a higher proliferation index grade, and a poorer prognosis in malignant soft tissue tumors. Our results also demonstrate the necessity of PD-L1 testing before starting PD-L1 checkpoint inhibitor therapy in soft tissue sarcomas. Larger clinical trials and longer follow-up are necessary to determine its function beyond that of a predictive biomarker.