Expression of PD-1 and PD-L1 in Kaposi’s Sarcoma and Their Association with Clinicopathologic Findings: A Retrospective Observational Study

doi:10.21203/rs.3.rs-8518462/v1

Background

Kaposi sarcoma (KS) is a multifocal vascular neoplasm that progresses from patch/plaque to tumor stage. Expression of PD-1 and PD-L1 is a key immune checkpoint feature in skin malignancies, but data on their role in KS are limited. We aimed to evaluate PD-1 and PD-L1 expression in KS and to assess their associations with pathologic stage and clinical features.

Methods

In this retrospective observational study, 46 patients with KS referred to Razi Hospital in Tehran were included. PD-1 and PD-L1 expression was evaluated by immunohistochemistry in tumor tissue and adjacent non-tumor tissue. Associations between marker expression, pathological stage, HIV status, and clinical relapse were analyzed.

Results

Intratumoral PD-L1 expression in tumor-stage samples was significantly higher than in patch/plaque-stage samples, while intratumoral PD-1 expression was significantly higher in patch/plaque lesions than in tumor-stage lesions. Similar stage-related differences were observed when a 5% cut-off for positivity was applied. In contrast, neither PD-1 nor PD-L1 expression in intratumoral or peritumoral areas showed a significant association with HIV status or clinical relapse.

Conclusion

Higher PD-L1 expression was associated with more advanced stages of KS, whereas PD-1 and PD-L1 expression was not related to HIV status or relapse. The upregulation of PD-1/PD-L1 expression suggests that anti-PD-1/PD-L1 immunotherapy may be a potential option for advanced KS.

Oncology

Kaposi sarcoma

Programmed cell death 1

Programmed death-ligand 1

Immune checkpoint inhibitors

Immunohistochemistry

HIV infection

Kaposi's sarcoma [1] is a multifocal vascular endothelial neoplasm with moderate biological potential, progressing from the patch/plaque stage to the nodule/tumor stage [2, 3]. About 10 to 20% of patients with classic KS will die, and a greater percentage will develop a secondary malignancy, which may be fatal [4]. KS has been reported as a rare cancer among the Iranian population based on the National Cancer Registry Organization [5]. A worse prognosis is usually seen in patients with visceral involvement, especially in the lungs [6].

Human herpesvirus 8 (HHV-8), the causative agent of KS, implicates the immune system in its pathogenesis. Immunomodulatory pathways may play an important role in the pathogenesis of disease, and antiretroviral therapy has been reported to resolve KS lesions [7, 8]. Distinguishing KS from other vascular tumors and other nonvascular spindle cell soft tissue neoplasms can sometimes be difficult. Therefore, latency-associated nuclear antigen (LANA) protein using immunohistochemistry (IHC) and detection of HHV-8 by polymerase chain reaction (PCR) are valuable for distinguishing KS from other lesions [9, 10].

In histopathological examination, this tumor is characteristically composed of large endothelial cells or spindle-shaped cells. The presence of extravasation of red blood cells, and infiltration of inflammatory mononuclear cells, macrophages, lymphocytes, and plasma cells also confirm the diagnosis [11]. Although the histopathology of the different clinical-epidemiologic forms of KS is essentially similar, the incidence, clinical presentation, and disease course differ markedly between classic, endemic, iatrogenic, and epidemic KS, reflecting both the background HHV-8 prevalence and the degree of immunosuppression [12–14]. From a clinical point of view, KS often appears as skin lesions that may occur anywhere on the body and may be flat, plaque-like, or nodular and vary in size. However, the absence of cutaneous Kaposi's lesions does not rule out visceral sarcoma [15].

Programmed Cell Death Protein 1 (PD-1) is a protein of the B7 family that is expressed on the surface of B and T lymphocytes and plays the role of regulating the immune system by suppressing its function. Programmed death-ligand 1 (PD-L1) is the ligand of PD-1 and plays a role in the negative regulation of the immune system. PD-1 and PD-L1 inhibitors are currently used in the treatment of some cancers [16]. Studies have shown that the expression of PD-1 and PD-L1 are associated with the clinical characteristics of various skin malignancies, including squamous cell carcinoma, melanoma, and NK/T-cell lymphoma [17–19]. PD-L1 expression causes dysfunction of the host's immune system, thereby facilitating the development of invasive warts [20]. PD-L1 expression is also increased in a subset of KS cases resistant to antiretroviral therapy, indicating the importance of PD-1/PD-L1 expression in its pathogenesis [21]. However, studies on PD-1/PD-L1 expression in KS are still limited and have reported heterogeneous results, often based on very small series and different scoring methods.

To address these gaps, we analyzed PD-1 and PD-L1 expression in KS cases using a compartmentalized approach, separately evaluating intratumoral and peritumoral regions and correlating expression with pathological stage and key clinical variables, including recurrence, HIV status, and HHV-8 infection. To our knowledge, this represents one of the larger series with detailed, quantitative assessment of PD-1/PD-L1 in KS.

In this study, we examined formalin-fixed paraffin-embedded tissue blocks from 46 KS patients referred to Razi Skin Hospital, Tehran, and extracted additional clinical data from their records. Clinical findings and pathological and immunohistochemical findings obtained from the pathology blocks of the patients were recorded by an experienced pathologist. PD-1 and PD-L1 expression was assessed using immunohistochemistry on tissue samples. The association between the expression status of PD-1 and PD-L1 markers with clinical features such as tumor stage and relapse status and HIV and HHV8 infection were evaluated. This study was conducted in line with the principles of the Declaration of Helsinki and according to the accepted regulations of the Tehran University of Medical Sciences ethical committee (IR.TUMS.MEDICINE.REC.1401.153). Informed consent was obtained from all participants.

Histology

The conventional classification of KS includes patch, plaque, or tumor stages. The patch stage is described by irregular proliferation of spindle cells with complex vascular channels and presence of the promontory sign. The plaque stage of KS is defined as increasing prominence of the features identified in the patch stage with limited cellular pleomorphism and a small number of mitoses. Finally, the tumor stage was defined by a well-defined nodular lesion with increased pleomorphism and prominent mitotic figures. Here in this investigation, the early stage was defined as the patch/ plaque stage and the advanced stage was defined as the tumor stage.

Immunohistochemistry

For immunohistochemical studies, formalin-fixed, paraffin-embedded tissue blocks were cut into 4-µm sections. Slides were stained with hematoxylin and eosin (H&E) for routine histologic evaluation. Immunohistochemistry for PD-1 and PD-L1 was performed on serial sections using commercially available monoclonal antibodies. After deparaffinization and rehydration, antigen retrieval was carried out according to the manufacturers’ recommendations. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Sections were then incubated with primary antibodies against PD-1 and PD-L1 for 30 minutes at room temperature. Immunoreactivity was detected with a standard polymer-based secondary detection system and visualized with 3,3'-diaminobenzidine (DAB) as chromogen, followed by hematoxylin counterstaining. Appropriate positive and negative controls were included in each run. The staining procedure followed previously published methods for PD-1/PD-L1 immunohistochemistry [17, 20].

Assessment of PD-1 and PD-L1 expression

In early-stage lesions, the intratumoral compartment was defined as the area composed of slit-like vascular spaces formed by spindle-shaped endothelial cells with cytologic atypia. In late-stage lesions, the intratumoral compartment corresponded to the well-circumscribed nodular spindle-cell proliferation. The peritumoral compartment was defined as the stroma immediately adjacent to, but outside, the tumor spindle-cell component. Two experienced pathologists independently delineated intratumoral and peritumoral regions and reviewed all slides in a blinded manner; any discrepancies were resolved by consensus.

For each case, PD-1 and PD-L1 expression were evaluated separately in intratumoral and peritumoral areas. PD-1 staining was scored mainly on lymphocytes and other mononuclear inflammatory cells showing membranous staining. PD-L1 staining was scored on tumor spindle cells and on mononuclear inflammatory cells within the tumor microenvironment, when present, considering membranous staining as positive. In each compartment, at least 100 cells were assessed across representative high-power fields, and the percentage of positively stained cells was visually estimated.

Two pre-specified cut-off values were used to define PD-1/PD-L1 “positivity.” The primary cut-off was ≥ 1% of cells showing specific staining, which is widely used in the literature as a minimal threshold. In addition, an exploratory cut-off of ≥ 5% was applied to evaluate whether a higher threshold produced different clinicopathologic associations, given variability in previously reported definitions. All subsequent analyses were performed using both the 1% and 5% cut-offs.

Statistical analysis

In the descriptive part, quantitative variables with mean and standard deviation and qualitative variables with number and percentage were reported. In the analytical part, the qualitative data were compared by the Fisher exact test or Chi-square. Quantitative data were analyzed from the perspective of normal distribution by Kolmogorov-Smirnov test and then compared by t-test. All analysis results will be with a significance level of 0.05 and at 95% confidence intervals. All statistical analyses were performed in SPSS version 22 software, and graphs related to statistical analyses were also drawn by this software. All analyses were conducted using both the 1% and 5% cut-off values for PD-1/PD-L1 positivity as defined above.

In this study, 46 tissue samples from patients with Kaposi’s sarcoma were evaluated. There were 33 men (71.7%) and 13 women (28.3%), with a mean age of 68.15 ± 15.12 years (range, 28–92 years). Based on clinical and histopathologic features, 23 lesions (50.0%) were classified as early patch/plaque stage and 23 lesions (50.0%) as advanced tumor stage. The lower limbs were the most common site of involvement (58.7%), followed by the upper limbs (26.1%). Overall, 6 cases (13.0%) showed clinical recurrence, and 4 patients (8.7%) were HIV-positive. Demographic and clinical characteristics according to lesion stage are summarized in Table 1.

Table 1. Demographic information of patients included in the study

Parameter		Total	patch/plaque	Tumor
Gender	Male	33 (71.7%)	17 (73.9%)	16 (69.6%)
Gender	Female	13 (28.3%)	6 (26.1%)	7 (30.4%)
Age (year)		68.15±15.12	68.15±15.2	—
Tumor location	Head and neck	5 (10.9%)	3 (13.0%)	2 (8.7%)
	Body	1 (2.2%)	1 (4.3%)	0 (0.0%)
	Upper limb	12 (26.1%)	4 (17.4%)	8 (34.8%)
	Lower limb	27 (58.7%)	15 (65.2%)	12 (52.2%)
	Perineum	1 (2.2%)	0 (0.0%)	1 (4.3%)
Recurrence	Yes	6 (13.0%)	2 (8.7%)	4 (17.4%)
Recurrence	No	40 (87.0%)	21 (91.3%)	19 (82.6%)
HHV8 status	Yes	7 (15.2%)	2 (8.7%)	5 (21.7%)
HHV8 status	Unknown	39 (84.8%)	21 (91.3%)	18 (78.3%)
HIV status	Yes	4 (8.7%)	3 (13.0%)	1 (4.3%)
HIV status	No	42 (91.3%)	20 (87.0%)	22 (95.7%)

Abbreviations: HHV-8, human herpesvirus 8; HIV, human immunodeficiency virus; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1.

Overall PD-1 and PD-L1 expression

The distribution of PD-1 and PD-L1 expression in intratumoral and peritumoral areas, using 1% and 5% cut-off points, is shown in Table 2. The mean percentage of PD-1 expression was 7.06 ± 13.50% in intratumoral areas and 18.5 ± 17.58% in peritumoral areas. The mean percentage of PD-L1 expression was 5.98 ± 9.34% intratumorally and 3.40 ± 6.11% peritumorally.

Using a 1% cut-off, intratumoral PD-1 expression was positive in 37/46 cases (80.4%), whereas peritumoral PD-1 expression was positive in 44/46 cases (95.7%). Intratumoral PD-L1 expression was positive in 27/46 cases (58.7%), and peritumoral PD-L1 in 19/46 cases (41.3%). Using a more stringent 5% cut-off, intratumoral PD-1 expression was positive in 20/46 cases (43.5%) and peritumoral PD-1 in 40/46 cases (87.0%). Intratumoral PD-L1 expression was positive in 20/46 cases (43.5%), while peritumoral PD-L1 expression was positive in 14/46 cases (30.4%) (Table 2). Representative examples of PD-L1–positive and PD-L1–negative lesions are shown in Figure 2A–C.

Table 2. Expression status of PD-1 and PD-L1 in intratumoral and peritumoral areas according to 1% and 5% cut-off points

cut-off point	Parameter	Positive	Negative
1%	Intratumoral PD-1 expression	37 (80.4%)	9 (19.6%)
	Peritumoral PD-1 expression	44 (95.7%)	2 (4.3%)
	Intratumoral PD-L1 expression	27 (58.7%)	19 (41.3%)
	Peritumoral PD-L1 expression	19 (41.3%)	27 (58.7%)
5%	Intratumoral PD-1 expression	20 (43.5%)	26 (56.5%)
	Peritumoral PD-1 expression	40 (87.0%)	6 (13.0%)
	Intratumoral PD-L1 expression	20 (43.5%)	26 (56.5%)
	Peritumoral PD-L1 expression	14 (30.4%)	32 (69.6%)

Abbreviations: PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1.

Association with tumor stage

At the 1% cut-off, intratumoral PD-L1 positivity was significantly more frequent in tumor-stage lesions than in patch/plaque lesions (18/23 [78.3%] vs 8/23 [34.8%]; p = 0.007). In contrast, there was no significant association between tumor stage and intratumoral PD-1 positivity (20/23 [87.0%] vs 17/23 [73.9%]; p = 0.459). Peritumoral PD-1 and PD-L1 positivity also did not differ significantly by stage (peritumoral PD-1: 22/23 [95.7%] vs 22/23 [95.7%], p = 1.000; peritumoral PD-L1: 7/23 [30.4%] vs 12/23 [52.2%], p = 0.231).

At the 5% cut-off, intratumoral PD-1 positivity was significantly more frequent in patch/plaque lesions than in tumor-stage lesions (16/23 [69.6%] vs 5/23 [21.7%]; p = 0.003). A representative example of intratumoral PD-1–positive mononuclear inflammatory cells is shown in Figure 3. Intratumoral PD-L1 positivity remained significantly higher in tumor-stage lesions compared with patch/plaque lesions (15/23 [65.2%] vs 6/23 [26.1%]; p = 0.017). Peritumoral PD-1 and PD-L1 positivity again showed no significant association with stage (peritumoral PD-1: 20/23 [87.0%] vs 19/23 [82.6%], p = 1.000; peritumoral PD-L1: 6/23 [26.1%] vs 7/23 [30.4%], p = 1.000) (Table 3).

Analysis of continuous expression percentages was consistent with these findings: the mean intratumoral PD-1 percentage was significantly higher in patch/plaque lesions than in tumor-stage lesions (p = 0.002), whereas the mean intratumoral PD-L1 percentage was significantly higher in tumor-stage lesions (p = 0.002). No significant stage-related differences were observed for mean peritumoral PD-1 or PD-L1 percentages (all p > 0.05).

Association with disease recurrence

We further evaluated the relationship between PD-1/PD-L1 expression and clinical recurrence (6 recurrent vs 40 non-recurrent cases) (Table 3).

At the 1% cut-off, intratumoral PD-1 was positive in 4/6 recurrent cases (66.7%) and 32/40 non-recurrent cases (80.0%; p = 0.598), and peritumoral PD-1 was positive in 6/6 (100%) vs 38/40 (95.0%; p = 1.000). Intratumoral PD-L1 was positive in 5/6 recurrent cases (83.3%) and 21/40 non-recurrent cases (52.5%; p = 0.212), while peritumoral PD-L1 was positive in 2/6 (33.3%) vs 16/40 (40.0%; p = 1.000). At the 5% cut-off, intratumoral PD-1 positivity was 3/6 (50.0%) in recurrent cases and 16/40 (40.0%) in non-recurrent cases (p = 0.680). Peritumoral PD-1 was positive in 4/6 (66.7%) vs 35/40 (87.5%; p = 0.221), intratumoral PD-L1 in 4/6 (66.7%) vs 16/40 (40.0%; p = 0.380), and peritumoral PD-L1 in 1/6 (16.7%) vs 11/40 (27.5%; p = 1.000).

Overall, there was no statistically significant association between PD-1 or PD-L1 expression and recurrence status, although intratumoral PD-L1 tended to be more frequently positive in recurrent cases.

Table 3. Association of the percentage and expression status of PD-1 and PD-L1 markers with tumor stage and disease recurrence

	Parameter	Tumor stage		P value	Recurrence		P value
	Parameter	Patch/plaque (23 cases)	Tumor (23 cases)	P value	Yes (6 cases	No (40 cases)	P value
Expression percentage	Intratumoral PD-1 expression	20 (87.0%)	17 (73.9%)	0.459	4 (66.7%)	32 (80.0%)	0.598
	Peritumoral PD-1 expression	22 (95.7%)	22 (95.7%)	1.000	6 (100.0%)	38 (95.0%)	1.000
	Intratumoral PD-L1 expression	8 (34.8%)	18 (78.3%)	0.007	5 (83.3%)	21 (52.5%)	0.212
	Peritumoral PD-L1 expression	7 (30.4%)	12 (52.2%)	0.231	2 (33.3%)	16 (40.0%)	1.000
Positive expression at 1% cut-off point	Intratumoral PD-1 expression	16 (69.6%)	5 (21.7%)	0.003	3 (50.0%)	16 (40.0%)	0.680
	Peritumoral PD-1 expression	20 (87.0%)	19 (82.6%)	1.000	4 (66.7%)	35 (87.5%)	0.221
	Intratumoral PD-L1 expression	6 (26.1%)	15 (65.2%)	0.017	4 (66.7%)	16 (40.0%)	0.380
	Peritumoral PD-L1 expression	6 (26.1%)	7 (30.4%)	1.000	1 (16.7%)	11 (27.5%)	1.000
Positive expression at 5% cut-off point	Intratumoral PD-1 expression	20 (87.0%)	17 (73.9%)	0.459	4 (66.7%)	32 (80.0%)	0.598
	Peritumoral PD-1 expression	22 (95.7%)	22 (95.7%)	1.000	6 (100.0%)	38 (95.0%)	1.000
	Intratumoral PD-L1 expression	8 (34.8%)	18 (78.3%)	0.007	5 (83.3%)	21 (52.5%)	0.212
	Peritumoral PD-L1 expression	7 (30.4%)	12 (52.2%)	0.231	2 (33.3%)	16 (40.0%)	1.000

Abbreviations: PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1.

Association with HIV status

The association between PD-1/PD-L1 expression and HIV infection is summarized in Table 4. Four patients (8.7%) were HIV-positive and 42 (91.3%) were HIV-negative. There were no statistically significant differences in mean intratumoral or peritumoral PD-1 or PD-L1 expression percentages according to HIV status (all p > 0.05).

At the 1% cut-off, intratumoral PD-1 was positive in 3/4 HIV-positive cases (75.0%) and 33/42 HIV-negative cases (78.6%; p = 0.869), and peritumoral PD-1 was positive in 4/4 (100%) vs 40/42 (95.2%; p = 0.655). Intratumoral PD-L1 was positive in 1/4 HIV-positive cases (25.0%) vs 25/42 HIV-negative cases (59.5%; p = 0.183), and peritumoral PD-L1 in 0/4 vs 18/42 (42.9%; p = 0.093). At the 5% cut-off, intratumoral PD-1 was positive in 2/4 HIV-positive and 17/42 HIV-negative patients (50.0% vs 40.5%; p = 0.712), while peritumoral PD-1 was positive in 3/4 (75.0%) vs 36/42 (85.7%; p = 0.569). Intratumoral PD-L1 was negative in all HIV-positive cases (0/4) and positive in 20/42 (47.6%; p = 0.066) HIV-negative cases, and peritumoral PD-L1 was negative in all HIV-positive cases (0/4) and positive in 12/42 (28.6%; p = 0.214) HIV-negative cases.

Taken together, these findings indicate that PD-1 and PD-L1 expression were not significantly associated with HIV status in this cohort, although HIV-positive patients tended to show lower rates of PD-L1 positivity, particularly in intratumoral areas.

Table 4. Association of the percentage and expression status of PD-1 and PD-L1 markers with HIV status

	Parameter	HIV		P value
	Parameter	Positive (4 cases)	Negative (42 cases)	P value
Expression percentage	Intratumoral PD-1 expression	7.23 ± 5.14	8.5 ± 61.4	0.665
	Peritumoral PD-1 expression	6.22 ± 7.21	6.17 ± 2.18	0.779
	Intratumoral PD-L1 expression	5.0 ± 25.0	7.9 ± 35.6	0.127
	Peritumoral PD-L1 expression	0	95.5 ± 21.3	0.149
Positive expression at 1% cut-off point	Intratumoral PD-1 expression	3 (75.0%)	33 (78.6%)	0.869
	Peritumoral PD-1 expression	4 (100.0%)	40 (95.2%)	0.655
	Intratumoral PD-L1 expression	1 (25.0%)	25 (59.5%)	0.183
	Peritumoral PD-L1 expression	0 (0.0%)	18 (42.9%)	0.093
Positive expression at 5% cut-off point	Intratumoral PD-1 expression	2 (50.0%)	17 (40.5%)	0.712
	Peritumoral PD-1 expression	3 (75.0%)	36 (85.7%)	0.569
	Intratumoral PD-L1 expression	0	20 (47.6%)	0.066
	Peritumoral PD-L1 expression	0	12 (28.6)	0.214

Abbreviations: HHV-8, human herpesvirus 8; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1.

In this retrospective observational study of KS patients, we observed stage-related differences in PD-1 and PD-L1 expression. Intratumoral PD-L1 expression was significantly higher in tumor-stage lesions than in patch/plaque-stage lesions, whereas intratumoral PD-1 expression was more prominent in early (patch/plaque) lesions. These patterns were consistent when expression was analyzed both as a continuous percentage and using 1% and 5% cut-offs for positivity. In contrast, peritumoral PD-1 and PD-L1 expression did not differ significantly according to histologic stage, and neither PD-1 nor PD-L1 expression in intratumoral or peritumoral compartments showed a significant association with HIV status or clinical recurrence. Taken together, these findings suggest that modulation of the PD-1/PD-L1 axis in KS is related mainly to tumor progression rather than to HIV infection or relapse status.

Immune checkpoint inhibitors targeting PD-1/PD-L1 have shown activity in several treatment-resistant malignancies. PD-L1 is reported to be expressed in approximately one-third of melanomas and in about half to two-thirds of cutaneous squamous cell carcinomas [22-25], and is often used as a predictive biomarker for response to anti–PD-1/PD-L1 therapy. Recent studies have also shown that PD-1/PD-L1 expression is significantly involved in the pathogenesis of benign skin warts and sarcomas [26, 27]. However, PD-1/PD-L1 interactions with clinical and pathological manifestations have not been sufficiently investigated in KS [28, 29]. Beyond individual case reports of checkpoint inhibition in KS, a recent multicenter phase II trial of pembrolizumab in classic and endemic KS reported an overall response rate of about 70%, with durable responses and acceptable toxicity, further supporting PD-1 blockade as a promising therapeutic option in this disease [30]. In this context, our data provide additional histopathologic evidence that PD-1/PD-L1 dysregulation is involved in KS biology and may be particularly relevant in advanced-stage lesions.

Previous studies evaluating PD-1/PD-L1 in KS have shown heterogeneous and controversial results. Chen et al. [31] reported strong PD-L1 expression in 60% of HIV-related KS patients. In another study, Mletzko et al [21] observed PD-L1 expression in 50% cases of HIV-associated KS. In contrast, a different study by Chen et al [32] found that all nine KS cases reviewed were negative for PD-L1 expression, whereas Paydas et al [33] reported PD-L1 positivity in four out of five KS cases (80%). The lack of standardization in cut-off values, antibody clones, and scoring criteria may contributes to this variability. Genovese et al. [34] demonstrated strong PD-L1 expression in classical KS (C-KS) on tumor-associated CD14+ cells, but not on spindle cells, using monochromatic staining for PD-L1 and CD14. Joest et al. [35] also showed stage-related changes in PD-L1 expression within the KS microenvironment. These observations suggest that both tumor cells and immune cells may contribute to PD-L1–mediated immune suppression in KS.

Kim et al. [30] examined PD-1 and PD-L1 expression according to pathological stage in 50 KS cases and reported stage-related differences, with higher PD-L1 expression in advanced lesions and higher PD-1 expression in early lesions, similar to our results. They also noted that these patterns were largely independent of clinical variables. In the present study, we used a compartmentalized approach and quantified PD-1 and PD-L1 expression separately in intratumoral and peritumoral areas. This approach allowed us to detect a prominent intratumoral PD-1 signal in early KS and increased intratumoral PD-L1 expression in tumor-stage lesions. This pattern is consistent with dynamic immune editing, in which early lesions show active immune infiltration, whereas more advanced lesions demonstrate upregulation of PD-L1 within the tumor, compatible with adaptive immune resistance.

These findings are in line with the current biological understanding of KS pathogenesis. KS is driven by HHV-8 infection, and disease progression reflects a balance between viral oncogenesis and host immune surveillance [36, 37]. In this model, infiltrating T cells recognize viral or tumor antigens and release interferon-γ, which then induces PD-L1 expression on both tumor cells and infiltrating myeloid cells in the tumor microenvironment [38, 39]. The result is a protective feedback loop for the tumor, in which PD-L1 upregulation inhibits T-cell activity and allows the tumor to escape immune destruction [38, 40]. This is consistent with our observation that nodular-stage KS, which likely reflects a more chronic inflammatory response, had higher PD-L1 levels. At the same time, early-stage lesions showed greater PD-1 expression on intratumoral lymphocytes, indicating an active immune response that may initially control tumor growth but can also lead to T-cell exhaustion [40, 41]. In addition, PD-1 expression has been shown to be upregulated on NK cells in patients with KS, where it is associated with reduced degranulation and IFN-γ production, further impairing tumor immune surveillance [36].

HHV-8 may also modulate the PD-1/PD-L1 axis directly. Host et al. reported that HHV-8 infection of monocytes upregulates PD-L1 and promotes the production of proinflammatory cytokines in a dose-dependent manner [42]. This suggests that the virus itself contributes to an immunosuppressive and tumor-promoting microenvironment [42, 43]. In our series, HHV-8 status was documented in a minority of cases, and the available data were not sufficient to draw firm conclusions about possible differences in PD-1/PD-L1 expression according to HHV-8 status or viral activity. Nevertheless, the combined evidence from our study and previous reports supports a model in which both virus-induced immune modulation and tumor-driven adaptive resistance converge on the PD-1/PD-L1 pathway in KS.

An important negative finding in our work is the absence of significant associations between PD-1/PD-L1 expression and either HIV status or clinical recurrence. Only 4 patients (8.7%) in our cohort were HIV positive, which reduces statistical power and may partially explain the lack of significant differences. It is also possible that, in the context of well-controlled HIV infection or classic KS, local factors within the tumor microenvironment have a stronger impact on PD-1/PD-L1 regulation than systemic HIV-related immune activation. Similarly, we did not observe a clear relationship between PD-1/PD-L1 expression and recurrence, although intratumoral PD-L1 tended to be more frequently positive in recurrent cases. Larger, longitudinal studies are needed to clarify whether checkpoint expression has prognostic value for KS outcomes.

This study has several strengths. It includes a relatively large number of KS cases compared with most previous immune checkpoint studies in this disease and uses a systematic, compartmentalized assessment of intratumoral and peritumoral expression. The use of two predefined cut-offs reflects thresholds commonly used in the literature and allowed us to confirm that the main stage-related findings were robust across different definitions of positivity. In addition, all slides were reviewed by experienced pathologists, and PD-1/PD-L1 scoring was performed in a blinded and reproducible manner.

Several limitations should also be acknowledged. First, the retrospective, single-center design introduces potential selection bias and limits control over treatment-related confounders, such as prior chemotherapy or radiotherapy, which may influence PD-1/PD-L1 expression. Second, the number of HIV-positive patients was small, which restricts our ability to draw firm conclusions regarding epidemic KS. Third, immunohistochemistry provides only a static snapshot of PD-1/PD-L1 expression; these markers are dynamic and can be modulated over time by changes in immune status and treatment, so a single time point may not fully capture their biological relevance. Finally, we focused only on PD-1 and PD-L1; more comprehensive profiling of other immune checkpoints and immune cell subsets was beyond the scope of this study but would provide a deeper characterization of the KS immune microenvironment.

In this study, PD-1 and PD-L1 expression in KS showed stage-related differences. Intratumoral PD-L1 expression was higher in advanced lesions, whereas intratumoral PD-1 expression was more prominent in early lesions. Neither PD-1 nor PD-L1 expression was associated with HIV status or clinical relapse. Given the limited efficacy of conventional chemotherapy in KS, our findings further support the evaluation of PD-1/PD-L1–directed immune checkpoint inhibitors as a therapeutic option for patients with advanced disease.

Ethics approval and consent to participate

This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Tehran University of Medical Sciences (approval code: IR.TUMS.MEDICINE.REC.1401.153). Written informed consent was obtained from all participants prior to inclusion in the study.

Consent for publication

Not applicable.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declares that there is no conflict of interest regarding the publication of this article. Funding

Not applicable.

Authors' contributions

SM collected all data and wrote the first draft of the manuscript. SM and NT analysed all data and interpreted the results. MHT and KKH were all involved in validation, critically review and editing of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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The authors declare no competing interests.

Expression of PD-1 and PD-L1 in Kaposi’s Sarcoma and Their Association with Clinicopathologic Findings: A Retrospective Observational Study

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Background

Materials and methods

Histology

Immunohistochemistry

Assessment of PD-1 and PD-L1 expression

Statistical analysis

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1