Skip to main content
NCBI home page
eBioMedicine logoLink to eBioMedicine
. 2017 Mar 24;18:50–55. doi: 10.1016/j.ebiom.2017.03.031

Programmed Death - Ligand 1 Expression Distinguishes Invasive Encapsulated Follicular Variant of Papillary Thyroid Carcinoma from Noninvasive Follicular Thyroid Neoplasm with Papillary-like Nuclear Features

Guodong Fu a,1, Olena Polyakova a,1, Christina MacMillan c,h, Ranju Ralhan a,b,c,d,e,h,⁎, Paul G Walfish a,b,d,e,f,g,⁎
PMCID: PMC5405181  PMID: 28363612

Abstract

Background

The noninvasive Encapsulated follicular variant of papillary thyroid cancer (EFVPTC) has been reclassified as Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) without a significant risk for malignant behavior. However the evaluation remains a challenge for clinicians. We sought to determine whether programmed death-ligand 1 (PD-L1) expression may serve as a biomarker to predict invasiveness of EFVPTC and assist to distinguish these neoplasms from NIFTP.

Methods

Immunohistochemical staining of PD-L1 expression was performed in sections of 174 Formalin-fixed paraffin-embedded (FFPE) tissue blocks from surgery removed thyroid nodules.

Results

Cytoplasmic PD-L1 expression was significantly increased in invasive EFVPTC (4.76 ± 1.49) as compared to NIFTP (3.06 ± 2.16, p < 0.001). Increased cytoplasmic PD-L1 expression was associated with invasiveness in EFVPTC (p < 0.001); PD-L1 positive EFVPTC cases were at 3.16 folds higher risk in developing invasion than the PD-L1 negative cases. No significant difference in cytoplasmic PD-L1 expression was observed between NIFTP and benign nodules.

Conclusion

PD-L1 expression may serve as a useful biomarker in predicting invasiveness of EFVPTC and distinguishing NIFTP from invasive EFVPTC. To our knowledge this is the first report suggesting the application of a protein biomarker to confirm NIFTP as benign indolent neoplasms.

Keywords: Thyroid cancer, Follicular variant, PTC, NIFTP, PD-L1, Invasion, Protein biomarker

Highlights

  • •

    Cytoplasmic PD-L1 levels were significantly lower in NIFTP as compared to EFVPTC.

  • •

    Cytoplasmic PD-L1 expression in NIFTP was similar to the benign subgroup.

  • •

    EFVPTC cases with higher cytoplasmic PD-L1 expression were at higher risk of capsular invasion.

The diagnosis of Encapsulated follicular variant of papillary thyroid cancer (EFVPTC) has become challenging and controversial. Follicular thyroid lesions currently have been reclassified as “noninvasive follicular thyroid neoplasms with papillary-like nuclear features” (NIFTP) versus capsular invasive EFVPTC using key histologic features to render these new diagnostic categories.

In this retrospective study, we observed significantly higher expression of Programmed Death-Ligand 1 (PD-L1) in invasive EFVPTC compared to NIFTP or benign subgroups. These observations suggest that PD-L1 may serve as a useful biomarker in predicting invasion in EFVPTC and distinguishing it from NIFTP.

1. Introduction

The global increasing incidence of thyroid cancer in part attributed to improved diagnostic tools and surveillance has culminated in over-diagnosis and over-treatment (Vaccarella et al., 2016). The follicular variant of papillary thyroid carcinoma (FVPTC) is a heterogeneous group of tumors, including the infiltrating and encapsulated variants (Shi et al., 2016). The diagnosis of encapsulated FVPTC (EFVPTC) without invasion as a malignant lesion remains controversial (Hodak et al., 2016, Nikiforov et al., 2016, Thompson, 2016). The microscopic diagnostic criteria for EFVPTC are follicular architecture, widespread nuclear features of papillary carcinoma, some additional features (papillae, fibrous bands, dense eosinophilic colloid), and the presence of capsule (Shi et al., 2016).

The noninvasive EFVPTC lesions having follicular growth pattern, particular microscopic architecture and nuclear features of PTC with low risk of malignancy have been reclassified as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) (Nikiforov et al., 2016, Strickland et al., 2016). The assessment of such borderline lesions is challenging for clinical practice as in this “grey zone” the intra-observer agreement may vary from 17% to 100% (Elsheikh et al., 2008). Recently Nikiforov et al. proposed diagnostic criteria for NIFTP which improved the overall diagnostic accuracy of NIFTP to 85.0–94.3% (Nikiforov et al., 2016). In EFVPTC, invasion rather than nuclear features influence tumor progression, patient management and prognosis (Ganly et al., 2015). Molecular testing may yield markers helpful in distinguishing invasive EFVPTC versus NIFTP where histological assessment of invasion is difficult or equivocal and ultimately may aid in differentiating these tumors by the fine needle aspirate (FNA) biopsy. Thus, ancillary methods to assess the invasive behavior of thyroid neoplasms are necessary to arrive at appropriate clinical decision to reduce the necessity of surgery and potential radioactive iodine (RAI) treatment. Biomarker-based ancillary screening may be a great asset in improving the diagnosis of NIFTP in pathology practice.

Recently, we reported programmed cell death ligand 1 (PD-L1) expression on tumor cells correlated with poor prognosis in thyroid cancer (Chowdhury et al., 2016). PD-L1 is a ligand for Programmed cell death 1 (PD-1/B7-H1) receptor to function as negative immune regulator. PD-1 is expressed on the surface of lymphocytes, such as T cells, B cells and natural killer T (NKT) cells. PD-L1 binding to PD-1 suppresses T-cell proliferation and cytokine release, such as IL-2 (Dong et al., 2002, Boussiotis, 2016). Its overexpression in a malignant neoplasm prevents malignant cells from being attacked by immune system due to a suppression of cytotoxic T cells. Thus, higher expression of PD-L1 in tumors will interfere with anti-tumor immunological attack and facilitate tumor growth and metastases. However, PD-L1 overexpression has not been examined to date to determine whether it can distinguish NIFTP from EFVPTC.

We hypothesized that PD-L1 expression could be a useful biomarker to distinguish NIFTP from invasive EFVPTC and thereby serve as to improve the accuracy of histopathological diagnosis of “grey zone” lesions which are in fact benign.

2. Materials and methods

2.1. Patients

This retrospective study was approved by Research Ethics Board (REB #07-0212-E) of Sinai Health System, Mount Sinai Hospital (MSH), a University of Toronto-affiliated hospital and a prime referral centre for thyroid diseases in Toronto, Canada. A total of 1859 cases of surgically removed thyroid nodules were carried out at Mount Sinai Hospital from Jan 2010 to Jun 2015. After analysis of clinical charts, surgical pathology reports and histological slides, 174 cases of surgically resected thyroid nodules were selected for this retrospective study using following inclusion criteria: the presence of a recorded chart with the gender and age of the patient at the time of diagnosis, recurrence episodes, size of the nodule, focality, area of fibrosis, capsular/vascular invasion, lymph node metastasis, as well as the existence of enough biological material in the paraffin blocks. Formalin-fixed paraffin-embedded (FFPE) tissue blocks from the selected 174 thyroid nodules were retrieved from MSH tumor bank. H&E stained sections were reviewed by two pathologists (CM, OP) and classified into four specific diagnostic subgroups- 52 NIFTPs and 45 cases of EFVPTC with invasion for comparison with 40 benign nodules and 37 cases of FVPTC with coexisting advanced lymphocytic or Hashimoto's thyroiditis (LT) (van Heerden et al., 1992, Nikiforov et al., 2012, Seethala et al., 2014, Nikiforov et al., 2016).

2.2. Inclusion/exclusion criteria

To identify NIFTP subgroup the inclusion criteria included major features (encapsulation/clear demarcation, follicular growth pattern, nuclear features of PTC: enlargement, crowding/overlapping, elongation, irregular contours, grooves, pseudoinclusions, chromatin clearing) and minor features (dark colloid, irregularly shaped follicles, intratumoral fibrosis, “sprinkling” sign, follicles cleft from stroma, multinucleated giant cells within follicles). The exclusion criteria for NIFTP were observed presence of “true” papillae > 1%, psammoma bodies, infiltrative border, tumor necrosis, high mitotic activity, cell/morphologic characteristics of other variants of PTC (Nikiforov et al., 2016).

To identify EFVPTC with invasion we used the features as encapsulated or well circumscribed nodule, follicular architecture, full thickness capsular invasion/vascular invasion or extrathyroidal extension, and 2–3 nuclear features of PTC using a 3-point nuclear scoring scheme (Nikiforov et al., 2016) (enlargement, crowding/overlapping, elongation, irregular contours, grooves, pseudoinclusions, chromatin clearing) (Seethala et al., 2014). Lymphocytic thyroiditis (LT) was separated from the FVPTC cases which did not satisfy the inclusion criteria for NIFTP and had no definite capsular or vascular invasion and had histological features of coexisting advanced lymphocytic or Hashimoto's thyroiditis. (van Heerden et al., 1992, Nikiforov et al., 2012, Seethala et al., 2014). All microcarcinomas (≤ 1 cm) were excluded from the study. Two pathologists independently reviewed all cases and separated them into 4 groups based on these inclusion/exclusion criteria.

2.3. Specimens and immunohistochemistry

FFPE tissue sections (5 μm thickness) were deparaffinized and rehydrated with series of graded alcohol. Antigen retrieval for PD-L1 was performed in Tris-EDTA buffer (pH = 9.0). After blocking the non-specific binding with background punisher (Biocare Medical, LLC, Concord, CA), the sections were incubated overnight (4 °C) with the monoclonal rabbit anti-PD-L1 antibody at 1:100 dilution (E1L3N, Cell Signalling Technology, Inc., Danvers, MA). The VECTASTAIN rapid protocol (Vector Labs, Burlington, ON, Canada) for immunostaining was performed. Diaminobenzidine (DAB) was used as the chromogen and counterstained with hematoxylin as described by us (Chowdhury et al., 2016). FFPE sections of classical variant of PTC were used as a positive control and histiocytes as internal positive controls. The thyroid cancer tissue incubated with an isotype specific IgG in place of the primary antibody was used as a negative control for PD-L1 staining in each batch of immunostaining.

2.4. Evaluation of immunostaining

Immunostaining scores were based on the percentage positivity and staining intensity as described previously (Chowdhury et al., 2016). Slides were scored as positive if epithelial cells showed immunoreactivity in the cytoplasm. Percentage positive scores were assigned according to the following scale: 0 ≤ 10%; 1 ≥ 11–30%; 2 ≥ 31–50%; 3 ≥ 51–70%; and 4 ≥ 71%. Staining intensity was scored semi-quantitatively as follows: 0 (none); 1 (mild); 2 (moderate) and 3 (intense) (Ralhan et al., 2015, Chowdhury et al., 2016). A total score was then obtained (ranging from 0 to 7) by adding the percentage positivity scores and intensity scores for each section. The IHC scoring was blinded from the histopathology report and was performed by two evaluators independently and used for subsequent analyses. The final score was given using following formula: [(percentage score 1 + intensity score 1) + (percentage score 2 + intensity score 2)]/2.

2.5. Statistical analysis

The data collected in a database designed for this study were analyzed using The SPSS 24.0 (SPSS, Chicago, IL, http://www.ibm.com/analytics/us/en/technology/spss/) program. A descriptive analysis of the population was performed. A multivariate analysis performed and the quantitative variables were described with central tendency measures: means, standard error and dispersion standard deviation, range to 95%. Means of PD-L1 scores were compared among the different diagnostic subgroups using one-way ANOVA test. The significance of the association between cytoplasmic PD-L1 positivity and invasiveness was examined by Fisher's exact test. The results were considered significant when the obtained 2-tailed values were p < 0.05.

3. Results

3.1. Patient characteristics and clinical-pathological parameters

The clinical and pathologic characteristics of the study cohort are summarized in Table 1. Among the 174 cases meeting the inclusion criteria, 40 cases were classified as benign nodules, 52 as NIFTP, 45 as EFVPTC with capsule or vessel invasion and 37 cases with lymphocytic thyroiditis (LT). Based on the staging classification system according to the 7th edition of American Joint Committee on Cancer (AJCC) (Edge and Compton, 2010), AJCC stages of NIFTPs and EFVPTCs were classified according to the pathology findings at resection as shown in Table 1.

Table 1.

Demographics and clinicopathologic features of patient cohort.

Characteristics Total cases (n = 174)
Age, mean (range) 50.2 (16–80)
Gender n (%)
 Male
 < 45 years 10 (6)
 ≥ 45 years 26 (15)
 Female
 < 45 years 50 (29)
 ≥ 45 years 88 (50)
Pathological diagnosis n (%)
 Benign
 Multiple hyperplastic nodules 34 (20)
 Cyst 4 (2)
 Follicular adenoma 2 (1)
 Neoplasm
 NIFTP 52 (30)
 EFVPTC 45 (26)
 Lymphocytic thyroiditis (LT)a 37 (21)
AJCC stages of neoplasmsb n (%)
 NIFTP
 Stage 0 2 (1)
 Stage I 31 (60)
 Stage II 12 (23)
 Stage III 5 (10)
 EFVPTC
 Stage I 27 (60)
 Stage II 13 (29)
 Stage III 5 (11)
Open in a new tab
a

Lymphocytic thyroiditis (LT) was separated from other categories because of coexisting advanced Lymphocytic or Hashimoto's thyroiditis.

b

AJCC staging for neoplasms of NIFTP and EFVPTC was based upon pathology findings at resection.

3.2. Cytoplasmic expression of PD-L1 is decreased in NIFTP

Immunohistochemical evaluation of cytoplasmic PD-L1 staining revealed significant differences in its expression between NIFTP and EFVPTC with invasion subgroups (Table 2). Increasing cytoplasmic PD-L1 immunostaining was observed from NIFTP (Fig. 1a & b) and benign nodules (Fig. 2a & b) to EFVPTC with invasion (Fig. 1c & d) and LT (Fig. 2c & d, Table 3). The negative and positive controls are shown in Fig. 3a & b, respectively. Multivariate analyses showed cytoplasmic PD-L1 expression was significantly increased in EFVPTC with invasion (mean ± standard deviation, 4.76 ± 1.49; 95% CI, 4.32–5.21) as compared to NIFTP (3.06 ± 2.16; 95% CI, 2.46–3.67, p < 0.001) as well as benign nodules (2.53 ± 1.34; 95% CI, 2.10–2.96, p < 0.001). There was no significant difference shown between benign nodules and NIFTP with p = 0.554. A significant increase in PD-L1 cytoplasmic expression was observed in EFVPTC with lymphocytic thyroiditis (4.51 ± 1.39; 95% CI: 0.79–3.31) comparing to benign (p < 0.001) as well as NIFTP subgroups (p < 0.001) (Table 2, Table 3). Based on cutoff point ≥ 4.5 for PD-L1 cytoplasmic score (sensitivity 91%, specificity 85%) (Chowdhury et al., 2016). 31 of 45 cases (69%) EFVPTC with invasion and 16 of 52 (31%) NIFTP cases were positive (Table 4). Fisher's exact test revealed that increased cytoplasmic PD-L1 expression statistically correlated with capsular invasion (p < 0.001). There was no strong association between cytoplasmic PD-L1 expression and age of patient, with a cut off 45 years old (p = 0.196, Fisher's exact test).

Table 2.

Comparison of cytoplasmic PD-L1 expression among subgroups of Benign, NIFTP, EFVPTC and Lymphocytic thyroiditis.

(I) Groups (J) Groups Mean difference (I–J) Std. error P value 95% Confidence interval
Lower bound Upper bound
Benign NIFTP − 0.61 0.0.41 0.554 − 1.79 0.58
EFVPTC − 2.33a 0.43 0.000 − 3.54 − 1.12
Lymphocytic thyroiditis − 2.05a 0.45 0.000 − 3.31 -0.79
NIFTP Benign 0.61 0.42 0.554 − 0.58 1.79
EFVPTC − 1.72a 0.36 0.000 − 2.73 − 0.71
Lymphocytic thyroiditis − 1.44a 0.38 0.003 − 2.51 -0.37
EFVPTC Benign 2.33a 0.43 0.000 1.12 3.54
NIFTP 1.72a 0.36 0.000 0.71 2.73
Lymphocytic thyroiditis 0.28 0.39 0.915 − 0.82 1.38
Lymphocytic thyroiditis Benign 2.01a 0.45 0.000 0.79 3.31
NIFTP 1.43a 0.38 0.003 0.37 2.51
EFVPTC − 0.28 0.39 0.915 − 1.38 0.82
Open in a new tab
a

The mean difference is significant at p < 0.05 level (ANOVA).

Fig. 1.

Fig. 1

Open in a new tab

Histology and immunohistochemical staining of PD-L1 expression in NIFTP and EFVPTC tissues.

(a), H&E stained tissue section of NIFTP showed follicular architecture. Insert: PTC cytological features: nuclear enlargement, grooves, micronuclei, pale to clear chromatin and crowding (thin black arrows). (b), Immunostaining of tissue section of NIFTP. Faint cytoplasmic PD-L1 immunostaining was detected in NIFTP. (c), H&E stained tissue section of EVFPTC showed the focal capsular invasion (thick black arrow), the follicular growth pattern of the tumor (T) as compared to normal thyroid (N). Insert: typical PTC cytological features: enlarged nuclei with central clearing, grooves and crowding (thin black arrows). (d), Immunostaining of tissue section of EFVPTC with capsular invasion. Moderate to strong cytoplasmic PD-L1 expression was observed in EFVPTC with invasion. Image magnifications are × 20 for (a) and (c), × 200 for (a) and (c) inserts, and × 400 for (b) and (d), respectively.

Fig. 2.

Fig. 2

Open in a new tab

Histology and immunohistochemical staining of PD-L1 expression in benign nodules and EFVPTC with lymphocytic thyroiditis tissues.

(a), H&E stained tissue section of benign hyperplastic nodule showed follicles of various sizes (thick black arrows). Insert: uniform round nuclei (thin black arrows). (b), Immunostaining of tissue section of benign neoplasm. No detectable cytoplasmic PD-L1 expression was observed in benign nodules. (c), H&E stained tissue section of Lymphocytic thyroiditis showed severe lymphoplasmacytic infiltration with germinal center formation and follicles (thick black arrows). Insert: extensive lymphocytic and plasmacytic infiltrate with oxyphilic metaplasia of follicular cells (thin black arrows). (d), Immunostaining of tissue section of EFVPTC with lymphocytic thyroiditis. Mild to moderate cytoplasmic PD-L1 expression was observed. Image magnifications are × 40 for (a) and (c), × 200 for (a) and (c) inserts, and × 400 for (b) and (d), respectively.

Table 3.

Cytoplasmic PD-L1 expression in subgroups of Benign, NIFTP, EFVPTC and Lymphocytic thyroiditis.

Subgroups N Mean ± SD 95% Confidence interval
Lower bound Upper bound
Benign 40 2.53 ± 1.34 2.10 2.96
NIFTP 52 3.06 ± 2.16 2.46 3.67
EFVPTC 45 4.76 ± 1.49 4.32 5.21
Lymphocytic thyroiditis 37 4.51 ± 1.39 4.04 4.97
Open in a new tab

Fig. 3.

Fig. 3

Open in a new tab

Negative and positive controls for immunohistochemical staining of PD-L1 expression.

(a), Negative control, a benign thyroid nodule incubated with isotype specific IgG in place of the anti-PD-L1 antibody did not show detectable immunostaining. (b), Positive control, an example of classic aggressive PTC section randomly selected for anti-PD-L1 antibody staining showed moderate to strong immunostaining. Image magnifications are × 400 for (a) and (b).

Table 4.

Comparison of cytoplasmic PD-L1 expression in NIFTP and EFVPTC.

Groups
 Total
NIFTP EFVPTC
PD-L1 cytoplasmic expression Negative cases 36 (69%) 14 (31%) 50
Positive cases 16 (31%) 31 (69%) 47
No. of total cases (%) 52 (100%) 45 (100%) 97
Open in a new tab

Notes: (1) Negative cases were defined by score of PD-L1 cytoplasmic expression < 4.5; Positive cases were defined by score of PD-L1 cytoplasmic expression ≥ 4.5.

(2) Fisher's exact test showed the positivity of cytoplasmic PD-L1 expression statistically associated with capsular invasiveness with p < 0.001.

The comparison of PD-L1 expression between benign subgroup and EFVPTC with invasion significantly correlated with invasion risk [Odds ratio (OR) 19.93, 95% CI: 1.59–3.07; p < 0.001]. The relative risk for cases with positive PD-L1 cytoplasmic expression was 3.16 folds higher than the PD-L1 negative cases.

4. Discussion

This study revealed cytoplasmic PD-L1 immunohistochemical analysis may serve as a potential ancillary method for distinguishing invasive EFVPTC from NIFTP and as an invasion predictor in EFVPTC to improve the accuracy of histopathological diagnosis of “grey zone” thyroid nodules thereby preventing patients from overtreatment and overdiagnosis. The four key novel findings are: (i) PD-L1 levels were significantly lower in NIFTP as compared to invasive EFVPTC; (ii) PD-L1 expression in benign and NIFTP subgroups was similar; (iii) EFVPTC cases with positive cytoplasmic PD-L1 expression were at higher risk of invasion; (iv) Lymphocytic infiltration enhanced PD-L1 expression.

To our knowledge, this is the first report to demonstrate the use of a protein biomarker to objectively distinguish NIFTP from EFVPTC with invasion. The pathological criteria recommended by Nikiforov et al. (2016) for identifying NIFTP are based on morphological features and adequate sampling of the tumor capsule interface to exclude invasion. The assessment of the whole capsule and evaluation of invasiveness could be challenging in some cases (e.g. sharp tumor bud invades into but not through the capsule, follicles aligned perpendicular, mushroom-shaped tumor within but not through the capsule, partially preserved capsule, FNA biopsy cytology features (Maletta et al., 2016). The use of a protein biomarker PD-L1 expression, combining with histopathology, will facilitate an accurate distinction between NIFTP and EFVPTC with invasion and thereby aid in selection of NIFTP patients for lobectomy without RAI or observation only, and invasive EFVPTC management by completion thyroidectomy ± RAI (Haugen et al., 2016). Recently, we reported increasing PD-L1 expression in thyroid cancer cells correlated with poor prognosis (Chowdhury et al., 2016). Upon binding to its specific receptor PD-1, it acts as an immune checkpoint regulator. This PD-L1/PD-1 pathway played a role in inhibiting T-cell receptor (TCR) mediated cell growth and cytokine production, resulting in peripheral tolerance of cancer cells and a failure to recognize and destroy the foreign cancer cells (Freeman et al., 2000, Blank et al., 2005, Okazaki and Honjo, 2007). Accordingly, the extent of PD-L1 expression on tumor cells largely emerges as a predictive biomarker in anti-PD-L1 directed cancer immunotherapy (Topalian et al., 2015). In the present study, we demonstrated that the expression of PD-L1 can be a predictor for distinguishing NIFTP from invasive EFVPTC.

Importantly, no significant difference in cytoplasmic PD-L1 expression between benign and NIFTP subgroups suggests that NIFTPs are more close to benign nodules, supporting the new paradigm for classification of NIFTP as non-malignant neoplasms (Maletta et al., 2016, Nikiforov et al., 2016, Patel, 2016, Thompson, 2016). The EFVPTC with invasion subgroup in which the cases were mainly classified as stage I and II at resection showed increased PD-L1 cytoplasmic staining. Using a completely different patient cohort in our previous study, a significant increase in both membrane and cytoplasmic PD-L1 expression was shown in patients with stage IV disease and correlated with high risk of aggressive disease, distant metastasis or death (Chowdhury et al., 2016). The correlation of cytoplasmic PD-L1 expression with risk of invasion in EFVPTC is in accord with our recent report of PD-L1 as a marker of thyroid cancer prognosis and aggressiveness (Chowdhury et al., 2016) and other cancers (Hamanishi et al., 2007, Leite et al., 2015). The cytoplasmic PD-L1 expression was significantly increased in EFVPTC with invasion comparing to NIFTP subgroup. These findings support the concept that NIFTP behaves primarily as a benign neoplasm. Based on several studies, including our previous report (Chowdhury et al., 2016), we classified cases of FVPTC without detectable capsular/vascular invasion and having coexisting diffuse lymphocytic infiltration into a separate LT subgroup since the mononuclear infiltration can influence the expression of PD-L1 and lead to a false positive result. The level of cytoplasmic PD-L1 expression was significantly higher in cases with lymphocytic infiltration than benign thyroid nodules, but similar to EFVPTC with invasion. This emphasizes that overexpression of PD-L1 in benign thyroid nodules should be interpreted with caution and that coexistence of chronic lymphocytic thyroiditis or Hashimoto's thyroiditis can cause a misleading false positive result (Taube et al., 2012, Cunha et al., 2013).

We are cognizant of the limitations that our findings are based on patients from a single clinical center; validation in independent patient cohorts is needed. Further, limited follow up period (1.5–5 years) did not permit a clear statement regarding the relationship of cytoplasmic PD-L1 overexpression with progression of invasive EFVPTC, their aggressiveness and/or recurrence.

In conclusion, examination of PD-L1 expression provided an add-on approach in distinguishing invasive EFVPTC from NIFTP and supported the concept that NIFTP is a non-malignant neoplasm. NIFTP patients showing high PD-L1 expression require follow up monitoring to exclude a risk of invasiveness. The level of PD-L1 expression may be useful in guiding their future management by improving the accuracy of histopathological diagnosis of “grey zone” thyroid nodules and pre-surgical cytological diagnosis thereby allowing a better selection of patients requiring surgery to prevent overtreatment and distress for patients.

Funding

The Mount Sinai Hospital Foundation of Toronto Da Vinci Gala Fundraiser, Mount Sinai Hospital Department of Medicine Research Fund, Alex and Simona Shnaider Chair in Thyroid Cancer (PGW) and Canadian Institutes of Health Research (CIHR) for CIHR Chair in Advanced Cancer Diagnostics (RR).

Disclosure statement

PGW and RR are shareholders in Proteocyte Diagnostics Inc. All the other authors (GF, OP and CM) have nothing to disclose.

Authors' contributions

RR and PGW conceptualized the study, contributed to the study design and to the manuscript. GF stained the slides, GF and OP conducted the experimental work. OP provided the clinical parameters and the follow-up data. GF and OP are equal contributors. CM performed the histopathology reporting of all the patients' tissues analyzed. OP and GF did the statistical analysis. RR and PGW provided the infrastructural support for this study. The manuscript was drafted by GF, OP and RR, and submitted for comments to all the authors. All authors approved the final version of the manuscript. RR and PGW are both senior and corresponding authors of this manuscript.

Acknowledgments

This study was supported by The Mount Sinai Hospital Foundation of Toronto Da Vinci Gala Fundraiser, Mount Sinai Hospital Department of Medicine Research Fund, Alex and Simona Shnaider Chair in Thyroid Cancer (PGW) and Canadian Institutes of Health Research (CIHR) for CIHR Chair in Advanced Cancer Diagnostics (RR). The funding sources had no role in the study design or its execution. The excellent technical assistance of Ronald S. Chazen in data analyses is gratefully acknowledged.

Presentations

This work was presented in part at the scientific program of the 86th Annual Meeting of the American Thyroid Association, Denver, Colorado, September 21–25, 2016.

Contributor Information

Ranju Ralhan, Email: Ranju.Ralhan@sinaihealthsystem.ca.

Paul G. Walfish, Email: Paul.Walfish@sinaihealthsystem.ca.

References

  1. Blank C., Gajewski T.F., Mackensen A. Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific T cells as a mechanism of immune evasion: implications for tumor immunotherapy. Cancer Immunol. Immunother. 2005;54(4):307–314. doi: 10.1007/s00262-004-0593-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boussiotis V.A. Molecular and biochemical aspects of the PD-1 checkpoint pathway. N. Engl. J. Med. 2016;375(18):1767–1778. doi: 10.1056/NEJMra1514296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chowdhury S., Veyhl J., Jessa F., Polyakova O., Alenzi A., MacMillan C., Ralhan R., Walfish P.G. Programmed death-ligand 1 overexpression is a prognostic marker for aggressive papillary thyroid cancer and its variants. Oncotarget. 2016;7(22):32318–32328. doi: 10.18632/oncotarget.8698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cunha L.L., Marcello M.A., Morari E.C., Nonogaki S., Conte F.F., Gerhard R., Soares F.A., Vassallo J., Ward L.S. Differentiated thyroid carcinomas may elude the immune system by B7H1 upregulation. Endocr. Relat. Cancer. 2013;20(1):103–110. doi: 10.1530/ERC-12-0313. [DOI] [PubMed] [Google Scholar]
  5. Dong H., Strome S.E., Salomao D.R., Tamura H., Hirano F., Flies D.B., Roche P.C., Lu J., Zhu G., Tamada K., Lennon V.A., Celis E., Chen L. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat. Med. 2002;8(8):793–800. doi: 10.1038/nm730. [DOI] [PubMed] [Google Scholar]
  6. Edge S.B., Compton C.C. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann. Surg. Oncol. 2010;17(6):1471–1474. doi: 10.1245/s10434-010-0985-4. [DOI] [PubMed] [Google Scholar]
  7. Elsheikh T.M., Asa S.L., Chan J.K., DeLellis R.A., Heffess C.S., LiVolsi V.A., Wenig B.M. Interobserver and intraobserver variation among experts in the diagnosis of thyroid follicular lesions with borderline nuclear features of papillary carcinoma. Am. J. Clin. Pathol. 2008;130(5):736–744. doi: 10.1309/AJCPKP2QUVN4RCCP. [DOI] [PubMed] [Google Scholar]
  8. Freeman G.J., Long A.J., Iwai Y., Bourque K., Chernova T., Nishimura H., Fitz L.J., Malenkovich N., Okazaki T., Byrne M.C., Horton H.F., Fouser L., Carter L., Ling V., Bowman M.R., Carreno B.M., Collins M., Wood C.R., Honjo T. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 2000;192(7):1027–1034. doi: 10.1084/jem.192.7.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ganly I., Wang L., Tuttle R.M., Katabi N., Ceballos G.A., Harach H.R., Ghossein R. Invasion rather than nuclear features correlates with outcome in encapsulated follicular tumors: further evidence for the reclassification of the encapsulated papillary thyroid carcinoma follicular variant. Hum. Pathol. 2015;46(5):657–664. doi: 10.1016/j.humpath.2015.01.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hamanishi J., Mandai M., Iwasaki M., Okazaki T., Tanaka Y., Yamaguchi K., Higuchi T., Yagi H., Takakura K., Minato N., Honjo T., Fujii S. Programmed cell death 1 ligand 1 and tumor-infiltrating CD8 + T lymphocytes are prognostic factors of human ovarian cancer. Proc. Natl. Acad. Sci. U. S. A. 2007;104(9):3360–3365. doi: 10.1073/pnas.0611533104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Haugen B.R., Alexander E.K., Bible K.C., Doherty G.M., Mandel S.J., Nikiforov Y.E., Pacini F., Randolph G.W., Sawka A.M., Schlumberger M., Schuff K.G., Sherman S.I., Sosa J.A., Steward D.L., Tuttle R.M., Wartofsky L. 2015 American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: the American Thyroid Association guidelines task force on thyroid nodules and differentiated thyroid cancer. Thyroid. 2016;26(1):1–133. doi: 10.1089/thy.2015.0020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hodak S., Tuttle R.M., Maytal G., Nikiforov Y.E., Randolph G. Changing the cancer diagnosis: the case of follicular variant of papillary thyroid cancer-primum non nocere and NIFTP. Thyroid. 2016;26(7):869–871. doi: 10.1089/thy.2016.0205. [DOI] [PubMed] [Google Scholar]
  13. Leite K.R., Reis S.T., Junior J.P., Zerati M., Gomes Dde O., Camara-Lopes L.H., Srougi M. PD-L1 expression in renal cell carcinoma clear cell type is related to unfavorable prognosis. Diagn. Pathol. 2015;10:189. doi: 10.1186/s13000-015-0414-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maletta F., Massa F., Torregrossa L., Duregon E., Casadei G.P., Basolo F., Tallini G., Volante M., Nikiforov Y.E., Papotti M. Cytological features of "noninvasive follicular thyroid neoplasm with papillary-like nuclear features" and their correlation with tumor histology. Hum. Pathol. 2016;54:134–142. doi: 10.1016/j.humpath.2016.03.014. [DOI] [PubMed] [Google Scholar]
  15. Nikiforov Y.E., Biddinger P.W., Thompson L.D.R. second ed. Lippincott Williams & Wilkins; 2012. Diagnostic Pathology and Molecular Genetics of the Thyroid: A Comprehensive Guide for Practicing Thyroid Pathology; pp. 152–182. [Google Scholar]
  16. Nikiforov Y.E., Seethala R.R., Tallini G., Baloch Z.W., Basolo F., Thompson L.D., Barletta J.A., Wenig B.M., Al Ghuzlan A., Kakudo K., Giordano T.J., Alves V.A., Khanafshar E., Asa S.L., El-Naggar A.K., Gooding W.E., Hodak S.P., Lloyd R.V., Maytal G., Mete O., Nikiforova M.N., Nose V., Papotti M., Poller D.N., Sadow P.M., Tischler A.S., Tuttle R.M., Wall K.B., LiVolsi V.A., Randolph G.W., Ghossein R.A. Nomenclature revision for encapsulated follicular variant of papillary thyroid carcinoma: a paradigm shift to reduce overtreatment of indolent tumors. JAMA Oncol. 2016;2(8):1023–1029. doi: 10.1001/jamaoncol.2016.0386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Okazaki T., Honjo T. PD-1 and PD-1 ligands: from discovery to clinical application. Int. Immunol. 2007;19(7):813–824. doi: 10.1093/intimm/dxm057. [DOI] [PubMed] [Google Scholar]
  18. Patel K.N. Noninvasive encapsulated follicular variant of papillary thyroid "cancer" (or not): time for a name change. JAMA Oncol. 2016;2(8):1005–1006. doi: 10.1001/jamaoncol.2016.0714. [DOI] [PubMed] [Google Scholar]
  19. Ralhan R., Veyhl J., Chaker S., Assi J., Alyass A., Jeganathan A., Somasundaram R.T., MacMillan C., Freeman J., Vescan A.D., Witterick I.J., Walfish P.G. Immunohistochemical subcellular localization of protein biomarkers distinguishes benign from malignant thyroid nodules: potential for fine-needle aspiration biopsy clinical application. Thyroid. 2015;25(11):1224–1234. doi: 10.1089/thy.2015.0114. [DOI] [PubMed] [Google Scholar]
  20. Seethala R.R., Asa S.L., Carty S.E., Hodak S.P., McHugh J.B., Richardson M.S., Shah J., Thompson L.D.R., Nikiforov Y.E. College of American Pathologists; Northfield, IL: 2014. Protocol for the Examination of Specimens From Patients With Carcinomas of the Thyroid Gland.http://www.cap.org/apps/docs/committees/cancer/cancer_protocols/2014/Thyroid_2014Protocol_3100.pdf [Google Scholar]
  21. Shi X., Liu R., Basolo F., Giannini R., Shen X., Teng D., Guan H., Shan Z., Teng W., Musholt T.J., Al-Kuraya K., Fugazzola L., Colombo C., Kebebew E., Jarzab B., Czarniecka A., Bendlova B., Sykorova V., Sobrinho-Simoes M., Soares P., Shong Y.K., Kim T.Y., Cheng S., Asa S.L., Viola D., Elisei R., Yip L., Mian C., Vianello F., Wang Y., Zhao S., Oler G., Cerutti J.M., Puxeddu E., Qu S., Wei Q., Xu H., O'Neill C.J., Sywak M.S., Clifton-Bligh R., Lam A.K., Riesco-Eizaguirre G., Santisteban P., Yu H., Tallini G., Holt E.H., Vasko V., Xing M. Differential clinicopathological risk and prognosis of major papillary thyroid cancer variants. J. Clin. Endocrinol. Metab. 2016;101(1):264–274. doi: 10.1210/jc.2015-2917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Strickland K.C., Vivero M., Jo V.Y., Lowe A.C., Hollowell M., Qian X., Wieczorek T.J., French C.A., Teot L.A., Sadow P.M., Alexander E.K., Cibas E.S., Barletta J.A., Krane J.F. Preoperative cytologic diagnosis of noninvasive follicular thyroid neoplasm with papillary-like nuclear features: a prospective analysis. Thyroid. 2016;26(10):1466–1471. doi: 10.1089/thy.2016.0280. [DOI] [PubMed] [Google Scholar]
  23. Taube J.M., Anders R.A., Young G.D., Xu H., Sharma R., McMiller T.L., Chen S., Klein A.P., Pardoll D.M., Topalian S.L., Chen L. Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci. Transl. Med. 2012;4(127):127ra137. doi: 10.1126/scitranslmed.3003689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thompson L.D. Ninety-four cases of encapsulated follicular variant of papillary thyroid carcinoma: a name change to noninvasive follicular thyroid neoplasm with papillary-like nuclear features would help prevent overtreatment. Mod. Pathol. 2016;29(7):698–707. doi: 10.1038/modpathol.2016.65. [DOI] [PubMed] [Google Scholar]
  25. Topalian S.L., Drake C.G., Pardoll D.M. Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell. 2015;27(4):450–461. doi: 10.1016/j.ccell.2015.03.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Vaccarella S., Franceschi S., Bray F., Wild C.P., Plummer M., Dal Maso L. Worldwide thyroid-cancer epidemic? The increasing impact of overdiagnosis. N. Engl. J. Med. 2016;375(7):614–617. doi: 10.1056/NEJMp1604412. [DOI] [PubMed] [Google Scholar]
  27. van Heerden J.A., Hay I.D., Goellner J.R., Salomao D., Ebersold J.R., Bergstralh E.J., Grant C.S. Follicular thyroid carcinoma with capsular invasion alone: a nonthreatening malignancy. Surgery. 1992;112(6):1130–1136. (discussion 1136–1138) [PubMed] [Google Scholar]

Articles from EBioMedicine are provided here courtesy of Elsevier

RESOURCES