Programmed Death Ligand 1 Expression in Lung Adenocarcinoma: An Analysis of the Histomorphological Features

Abstract

Background:

Immunotherapy is now a vital target therapy in the advanced cases of lung adenocarcinoma. The outstanding result of therapies with medications that inhibit the interaction of programmed death ligand 1 with programmed death protein 1 has revolutionarized prognostic treatment regimes.

Aims:

The study was undertaken with the objectives to analyze the detailed histomorphological features of adenocarcinoma lung with programmed death ligand-1 (PD-L1) expression in tumor cells and to compare the histomorphological features with PD-L1 negative group.

Materials and Methods:

The present study is a retrospective case series with 100 cases of non-small cell lung cancer-adenocarcinoma phenotype in which testing for PD-L1 had been done using immunohistochemistry. Detailed histomorphological analysis and comparison was performed for both the PD-L1 positive and negative phenotype.

Results:

Histomorphological features of 25 cases with positive PD-L1 positivity in the tumor cells and 75 cases that were negative for PD-L1 were analyzed. The most frequent pattern in the category that was PD-L1 positive was singly scattered cells or loose clusters present in 84% cases followed by solid nests that was identified in 60% cases. The presence of solid nests in the PD-L1 positive category was statistically significant (P = 0.018). Mucin was identified in 24% cases, and tumor necrosis was documented in 60% cases with PD-L1 positivity. In the cluster that was PD-L1 positive, 92% cases had moderate-to-severe nuclear pleomorphism.

Conclusion:

The identification of histomorphological patterns and characteristics may aid in triaging cases that have the likelihood to harbor PD-L1-positive phenotype, which has predictive and prognostic outcome.

INTRODUCTION

Worldwide, in most developed countries, the leading cause of cancer deaths is lung malignancy. In spite of multidisciplinary therapies administered in patients with metastatic or advanced non-small cell lung cancer (NSCLC), the overall survival is generally dismal.[1] The approach to the treatment and molecular diagnostics of NSCLC-adenocarcinoma phenotype has progressed significantly in the last decade.[2] The development of medications that target precise sub-categories of tumors identified by their protein expression or genetic morphology has radicalized therapeutics.[3,4] Immunotherapy is now an important target therapy in advanced cases of NSCLC. The outstanding result of therapies with medications that inhibit the interaction of programmed death ligand 1 with programmed death protein 1 has revolutionarized prognostic treatment regimes.[5,6]

The International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society has categorized adenocarcinoma according to its major growth pattern including acinar, solid, papillary, micropapillary, and lepidic subtypes; and variants including mucinous, colloid, enteric, and fetal types have been enlisted.[7] The correlations between various molecular mutations and histologic sub-types have been reported in NSCLC-adenocarcinoma.[8] Programmed death ligand-1 (PD-L1) positivity in adenocarcinoma phenotype is related with advanced disease and lymph node involvement.[9] A detailed analysis of the histomorphological features may define the presence of specific pattern or feature in the phenotype with PD-L1 expression. The study was undertaken with the objectives to analyze the detailed histomorphological features of adenocarcinoma lung with PD-L1 expression in tumor cells and to compare histomorphological features with PD-L1 negative group.

MATERIALS AND METHODS

The present study is a retrospective case series with 100 cases of NSCLC adenocarcinoma phenotype in which testing for PD L1 had been done using immunohistochemistry. The study had been approved by the Institutional Ethical Committee. (IEC 6/16 and 12/17). Written informed consent was taken from all cases that have been included in the present study. The testing was done using the anti PD-L1 monoclonal antibody clone SP-263 from Ventana medical systems (USA) using the Optiview (DAB) detection kit on the Ventana benchmark XT automated staining system. A positive control (human placental tissue) was run with very batch. Cytoplasmic/membranous staining in the tumor cells was assessed. Cases with >1% staining in the tumor cells were categorized as positive. The study included 100 cases out of which 25 cases had positive expression of PD-L1 in the tumor cells, whereas 75 cases were negative for PD-L1. The archival hematoxylin and eosin-stained slides were retrieved from the records. Detailed analysis of the histomorphological features was done. The histomorphological analysis was done using parameters including the cellular pattern of arrangement-solid nests, acinar pattern, papillary/micropapillary architecture, lepidic pattern, and presence of loose cohesive clusters or singly scattered cells. The nuclear pleomorphism was assessed as mild, moderate, or severe. The presence of intracellular or extracellular mucin was documented. The presence or absence of tumor necrosis was noted. The tumors were categorized based on their differentiation into well, moderate, or poorly differentiated lesions. This detailed histomorphological analysis was performed for both the PD-L1 positive and negative phenotype.

Statistical analysis

IBM-Statistical Package for the Social Sciences (SPSS, International Business Machines Corporation, New York, USA) analysis software, version 16 was used for the statistical analysis. The histomorphological features of cases with positive PD-L1 expression were analyzed and compared with cases that were negative for PD-L1. The categorical variables were evaluated using the Chi-square test. Two-sided tests were used for the calculation of P value. P ≤ 0.05 was considered statistically significant while P ≤ 0.01 was highly significant.

RESULTS

The present study is a retrospective case series that comprised 100 cases of NSCLC-adenocarcinoma phenotype including 25 cases with positive PD-L1 positive expression in the tumor cells and 75 cases that were negative for PD-L1.

Cellular pattern of arrangement

A detailed analysis of cellular pattern revealed the most frequent pattern in the phenotype expressing PD-L1 was singly scattered cells or loose clusters present in 84% (n = 21) cases followed by solid nests that was identified in 60% (n = 15) cases [Figure 1a-c]. The most uncommon pattern in cases that were positive for PD-L1 was the lepidic pattern present in 4% (n = 1) cases followed by the micropapillary or papillary pattern that was present in 8% (n = 2) cases. In the PD-L1 negative group, the most frequent pattern was also singly scattered cells or loose clusters documented in 81.33% (n = 61) cases followed by the acinar pattered that was identified in 57.33% (n = 43) cases [Figure 1d]. The solid pattern was identified in only 33.33% cases in the category that was PD-L1 negative. As comparison of the histomorphological patterns between the two categories revealed that the presence of solid nests in the PD-L1 positive cluster was statistically significant (P = 0.018) as opposed to the PD-L1 negative group [Table 1].

Figure 1.

Morphological patterns: (a): Tumour cells arranged in solid nests, (b and c): Tumour cells disposed in loose clusters along with singly scattered cells, (d): Acinar pattern along with presence extracellular mucin, (e): Tumour cells arranged in loose clusters along with presence of necrosis, (f): Tumour cells with severe nuclear pleomorphism and presence of atypical mitoses (a-d: H and E, ×100), (e and f: H and E, ×200)

Table 1.

Comparison of the histomorphological characteristics of the programmed death ligand 1 positive group versus the programmed death ligand 1 negative group

Histomorphological characteristics	Total (n=100)	PDL1 positive (n=25), n (%)	PDL1 negative (n=75), n (%)	P
Cellular pattern of arrangement
Solid pattern
Present	40	15 (60)	25 (33.33)	0.018*
Absent	60	10 (40)	50 (66.66)
Acinar pattern
Present	54	11 (44)	43 (57.33)	0.246
Absent	46	14 (56)	32 (42.66)
Papillary/micropapillary pattern
Present	15	2 (8)	13 (17.33)	0.257
Absent	85	23 (92)	62 (82.66)
Lepidic pattern
Present	12	1 (4)	11 (14.66)	0.155
Absent	88	24 (96)	64 (85.33)
Singly scattered cells/loose clusters
Present	82	21 (84)	61 (81.33)	0.763
Absent	18	4 (16)	14 (18.66)
Nuclear pleomorphism
Mild	19	2 (8)	17 (22.66)	0.105
Moderate or severe	81	23 (92)	58 (77.33)
Tumour differentiation
Well	16	1 (4)	15 (20)	0.126
Moderate	54	14 (56)	40 (53.33)
Poor	30	10 (40)	20 (26.66)
Mucin (extracellular or Intracytoplasmic)
Present	19	6 (24)	13 (17.33)	0.461
Absent	81	19 (76)	62 (82.66)
Necrosis
Present	45	15 (60)	30 (40)	0.081
Absent	55	10 (40)	45 (60)

PDL1: Programmed death ligand 1

Mucin

The presence of intracellular or extracellular mucin was assessed in both the PD-L1 positive and negative categories [Table 1]. Mucin (intracellular or extracellular) was identified in 24% (n = 6) cases in the category that was PD-L1 positive. In contrast, only 17.33% (n = 13) in the PD-L1 negative group demonstrated the presence of mucin [Figure 1d]. An assessment of the PD-L1 positive and negative phenotypes in terms of presence of intracellular or extracellular mucin was not statistically significant.

Necrosis

The presence of tumor necrosis was documented in 60% (n = 15) cases in the PD-L1 positive category [Figure 1e]. In the PD-L1 negative cluster, necrosis was identified in only 40% (n = 30) cases [Table 1]. A statistically comparison of this parameter between the PD-L1 positive and negative phenotypes was insignificant.

Nuclear pleomorphism

In the PD-L1 positive group, 92% (n = 23) cases had moderate-to-severe nuclear pleomorphism, whereas only 2 cases (8%) had mild nuclear pleomorphism. In the PD-L1 negative phenotype, 22.66% (n = 17) cases had mild nuclear pleomorphism while 58 cases (77.33%) cases demonstrated moderate-to-severe nuclear pleomorphism [Figure 1f]. A comparison of the PD-L1 positive versus PD-L1 negative groups based on nuclear pleomorphism was not statistically significant [Table 1].

Tumor differentiation

In both the PD-L1 positive and PD-L1 negative categories, the tumors were categorized as well, moderate or poorly differentiated lesions. In cases with the PD-L1 positive phenotype, 56% (n = 14) were moderately differentiated, whereas 40% (n = 10) were poorly differentiated. Well-differentiated tumors comprised of only 4% (n = 1) cases in the group expressing PD-L1. In contrast, in the PD-L1 negative cluster, 20% (n = 15) cases were well differentiated and only 26.66% (n = 20) cases were poorly differentiated [Table 1]. A evaluation of tumor differentiation among the PD-L1 positive and negative phenotypes was not statistically significant.

DISCUSSION

The current study is a novel attempt to define the specific histomorphological features of in cases of NSCLC-adenocarcinoma subtype with PD-L1 expression in the tumor cells. The most frequent pattern in the PD-L1 positive category was the presence of loose cohesive clusters or singly scattered tumor cells followed by the solid pattern. In the study conducted by Zhang et al., the authors stated that the PD-L1 expression is possibly chiefly related to the solid phenotype.[10] Jin et al. concluded from their study that solid and micropapillary patterns of cellular arrangement are more likely to be related to PD-L1 positivity.[2] Miyazawa et al. concluded from their study that the solid predominant adenocarcinoma is more frequently express PD-L1 when compared with the other sub-types.[1] Lepidic and acinar patterns are the least common patterns associated with PD-L1 positivity.[11,12] This finding was concordant with the current study, wherein in the PD-L1 positive category, lepidic pattern was the most uncommon. Driver et al. concluded from their study that the subtype groups in adenocarcinoma lung with least frequent PD-L1 expression were acinar and papillary subtypes.[13]

In the current study, the presence of solid pattern was consistent in cases with the PD-L1 phenotype and was significant statistically when equated with the PD-L1 negative groups. This result is concordant with the outcomes of the research conducted by Phruttinarakorn et al. wherein the authors concluded that solid predominant adenocarcinoma was constantly related with high PD-L1 positivity.[9] The results of the research studies conducted by Driver et al. and Pan et al. displayed substantially higher levels of PD-L1 expression in the solid types of adenocarcinoma lung when compared with the nonsolid subtypes.[13,14] In the genetic and immune profile study conducted by Dong et al., that included 194 cases of NSCLC-adenocarcinoma phenotype demonstrated that the solid subtype is significantly related to tumor mutation burden and presence of adaptive immune resistance that is associated with the benefits of administering PD-1/PD-L1 inhibitors in PD-L1 positive solid type adenocarcinoma.[15] Igarashi et al. estimated PD-L1 positivity in various subcategories of adenocarcinoma lung and stated that there were no significant differences among the various sub-types.[16]

The presence of mucin (intracytoplasmic or extra-cytoplasmic) was identified in 24% cases in the present study among the PD-L1-positive category. In the study conducted Driver et al., mucinous morphology was identified in 27% cases with PD-L1 positive phenotype and was one of the least common sub-types.[13] In the current study, there was no statistical significance with the presence of mucin and PD-L1 expression.

Tumor necrosis was identified in 60% cases in the PD-L1 positive group as compared to 40% in the PD-L1 negative group in the present study. Although this finding is not statistically significant, the presence of necrosis implies high tumor multiplication. Necrosis is generally a feature of high-grade tumors with significant tumor mutational burden.[17-19]

In the present study, 92% cases in the PD-L1 positive cluster demonstrated moderate to severe nuclear pleomorphism. Majority of the tumors (96%), in the category expressing PD-L1 were moderately to poorly differentiated lesions on histology. This result is concordant with the outcomes of the study done by Driver et al., wherein the researchers stated that PD-L1 positivity was related with higher histologic grade.[13] In the meta-analysis conducted by Wang et al. that included 1157 patients from 6 studies, the authors stated the PD-L1 positivity was significantly related with poorly differentiated tumors and resulted in poor overall survival.[20] Jin et al. concluded from their study, that in cases of NSCLC-adenocarcinoma phenotype, the well-differentiated tumors had a lower frequency of PD-L1 expression when compared with the poorly differentiated lesions.[2] In contrary to the findings of the current study, Velcheti et al. described that the results of PD-L1 phenotype are independent of histology for cases of NSCLC-adenocarcinoma subtype.[21]

A limitation of the study is that there is no correlation between the various histomorphological patterns and the survival benefits. However, the current study aimed to define the various histologic subtypes in the PD-L1 positive and negative clusters and identify any specific morphology in the PD-L1 positive phenotype.

CONCLUSION

The current study is an innovative attempt to try and define the specific histomorphological features and patterns in NSCLC-adenocarcinoma with PD-L1 positive expression. The cases with PD-L1 positive phenotype show presence loose clusters, singly scattered cells or solid patterns of cellular arrangement along with presence necrosis. Majority of tumors with PD-L1 positivity have moderate to severe nuclear morphology along with higher tumor grade. The identification of histomorphological patterns and characteristics may aid in triaging cases that have the likelihood to harbor PD-L1 positive phenotype, which has predictive and prognostic outcome.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.