Keywords
PD-L1 - immunohistochemistry - sarcomas - immunotherapy - immune checkpoint inhibitors
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]
[3]
[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]
[6]
[7]
[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]
[10]
[11]
[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.
Aims and Objectives
In the present research, we studied the immunohistochemical expression of PD-L1 and
the Ki-67 proliferation index in various soft tissue sarcomas and their correlation
with clinicopathological parameters.
Materials and Methods
This was a prospective study done for the duration of 1 year (November 2019–October
2020) in the Department of Pathology, 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
biopsy- and 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 (≥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.
Statical Analysis
Data were analyzed using Statistical Package for Social Sciences (SPSS, IBM Inc.,
United States), version 21.0. The independent sample t-test for continuous variables and the chi-square test for discrete variables were
used to investigate the relationships between PD-L1 expression and clinicopathologic
factors. A p-value less than 0.05 was considered statistically significant.
Results
Demographic Profile of the Study Population
The final study population included 50 cases of soft tissue sarcomas. The age range
of cases enrolled in the study was from 2 to 70 years. The mean age of the study population
was 34.26 ± 18.46 years. The vast majority of the patients (64%) (32/50) were between
the ages of 21 and 60 years, followed by 30% (15/50) younger than 20 years, and 6.0%
(3/50) of cases were aged >60 years. The majority of cases enrolled in the study were
males: 68% (34/50) and 32% (16/50) were females. The male-to-female ratio was 2.13:1.
Distribution of the Study Population According to Histological Grade, Proliferation
Index Grade, and Sarcoma Subtypes
Among the study population, 52% (26/50) of patients were in FNCLCC grade II, followed
by 30% (15/50) in grade III and 18% (9/50) patients in grade I; 38% (19/50) of patients
were in proliferation index grade II, followed by 34% (17/50) in grade III and 28%
(14/50) patients in grade I. Among soft tissue malignant neoplasms, 36% (18/50) cases
were of synovial sarcoma, followed by 18% (9/50) cases of Ewing's sarcoma, 12% (6/50)
of dermatofibrosarcoma protuberans (DFSP), 10% (5/50) liposarcoma, 10% (5/50) leiomyosarcoma,
8% (4/50) undifferentiated pleomorphic sarcoma and 6% (3/50) of rhabdomyosarcoma.
Association of PD-L1 Expression with Proliferation Index Grade
Proliferation index grade III accounts for 62.5% (10/16) of cases with positive PD-L1
expression, followed by proliferation index grade II with 25% (4/16) cases and grade
I with 12.5% (2/16) cases. On comparing statistically, this difference was found to
be significant (p = 0.013) ([Table 1]).
Table 1
Association of PD-L1 with proliferation index grading
|
Proliferation index grade
|
PD-L1 ≤ 1% (n = 34)
|
PD-L1 > 1%(n = 16)
|
|
Number
|
Percentage (%)
|
Number
|
Percentage (%)
|
|
Grade I (n = 14)
|
12
|
35.29
|
2
|
12.5
|
|
Grade II (n = 19)
|
15
|
44.12
|
4
|
25.0
|
|
Grade III (n = 17)
|
7
|
20.59
|
10
|
62.5
|
Abbreviation: PD-L1, programmed death ligand-1.
Note: Chi-square = 8.686 (df = 2); p = 0.013.
Association of PD-L1 Immunohistochemical Expression with Histological Grading, Histopathological
Diagnosis, and Prognosis
The majority (56.25%) (9/16) of cases with positive PD-L1 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.
Note: 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]) ([Figs. 1] and [2]).
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.
Note: Chi-square = 13.057 (df = 6); p = 0.042,
Fig. 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 ×400); (C) dermatofibrosarcoma protuberans; (D) positive expression of PD-L1 in tumor cells of dermatofibrosarcoma protuberans (PD-L1
×400); (E) Ewing's sarcoma; (F) positive expression of PD-L1 in tumor cells of Ewing's sarcoma (PD-L1 ×400). PD-L1,
programmed death ligand-1.
Fig. 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 ×400); (C) undifferentiated pleomorphic sarcoma; (D) positive expression of PD-L1 in tumor cells of undifferentiated pleomorphic sarcoma
(PD-L1 ×400); (E) leiomyosarcoma; (F) positive expression of PD-L1 in tumor cells of leiomyosarcoma (PD-L1 ×400). PD-L1,
programmed death ligand-1.
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 PD-L1 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.
Note: 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 tissue sarcoma,
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]
[24]
[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]). The reported 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.
Table 5
Comparison of PD-L1 positivity among soft tissue sarcomas in different studies
|
Name of author (y)
|
Number of cases included in study
|
Criteria for positivity of PD-L1
|
Number (%) of PD-L1 positive cases
|
|
Orth et al[14] (2020)
|
247
|
1%
|
39 (15.8%)
|
|
Kösemehmetoğlu et al[23] (2017)
|
222
|
5%
|
34 (15%)
|
|
Kim et al[11] (2016)
|
82
|
10%
|
35 (43%)
|
|
Paydas et al[24] (2016)
|
65
|
5%
|
19 (29%)
|
|
D'Angelo et al[13] (2015)
|
50
|
1%
|
6 (12%)
|
|
Present study (Mishra et al, 2023)
|
50
|
1%
|
16 (32%)
|
Abbreviation: PD-L1, programmed death ligand-1.
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]
[27]
[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 PD-L1 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.
Conclusion
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.
