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Inflammation in the neoplasms of the adrenal gland: Is there a prognostic role? An immunohistochemical study
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Elia Guadagno*, Daniela Russo, Sara Pignatiello, Marialaura Del Basso De Caro Department of Advanced Biomedical Sciences, Pathology Section, Federico II University of Naples, Via Pansini, 5- 80131, Naples, Italy
ARTICLE INFO
Keywords:
Adrenal gland tumors Inflammation
CD3
PDL-1
CD45
CD20
ABSTRACT
Introduction: Adrenal gland neoplasms are mostly benign. The differential diagnosis between adrenocortical adenoma and carcinoma relies on nine morphologic parameters (Weiss criteria) that are mostly subjective. Although rare, carcinomas represent an aggressive disease that require short time follow-up. For this reason, the diagnosis should be accurate. Neoplasms of the medulla are mostly represented by phaeochromocytomas, all potentially metastatic. Prognostic score systems (GAPP and PASS) have been implemented but not enough objective and useful in borderline cases. More objective parameters should be introduced. Little is known in literature on the inflammatory response in these tumors. Aim of our study was the definition (type, density and distribution) of inflammation in the adrenal neoplasms.
Material and methods: Immunohistochemistry for CD45 (inflammatory cells), CD20 (B cells) and CD3 (T cells) antibodies was performed in 15 adrenocortical neoplasms and 17 phaeochromocytomas. A manual count of the signal was set for each marker, to establish the cellular type, their density (cells/mm2) and location within the tumor. Fisher’s exact test was applied to assess the correlation between the immunoscore and clinico-pathologic parameters.
Results: The difference of cellular density between the three markers was statistically significant (p value = 0.0028), with highest values for CD45 and CD3. No differences were detected between the periphery and the center of the lesions. The most relevant finding was the detection of a higher immunoscore in adrenocortical adenomas, compared to carcinomas. Moreover, most of phaeochromocytomas showed high expression of in- flammation, except the only metastatic case.
Conclusions: The present study showed that inflammation could represent a valuable diagnostic and potential prognostic parameter, useful for the correct management of these lesions.
1. Introduction
The adrenal gland is a special type of endocrine organ because of its double nature, consisting of the cortex and the medulla, two parallel districts that differ in structure, function and development.
The cortex is furtherly subdivided into three layers (the zona glo- merulosa, the fasciculata and the reticularis), each one with a specific hormonal identity. In all the three sublayers. Two main types of tumors can originate, a benign form, the adenoma, and a malignant form, the carcinoma. The adrenal cortical adenomas (ACA) are common, af- fecting 10 % [1] of the population and mostly representing an in- cidental finding. They can be non-functioning or associated to en- docrinopathies, based on the tumor hormonal secretory activity. The diagnosis is based on clinical and hormonal evaluation, besides dedi- cated adrenal imaging to assess lipid content [2]. Adrenal cortical
carcinomas (ACC) are rare neoplasms [3], with an annual incidence of 0.5-2 cases per 1 million population. They can be hormonally func- tioning in a half of cases (generally associated to hypercortisolism), they can present with symptoms related to tumor growth in a third of cases (pain and abdominal fullness) or they can be completely asymp- tomatic in other cases. ACC represent an aggressive disease, with a 5- year survival rate between 37 and 47 %. They can be subdivided into a low grade and a high-grade form, based on the mitotic activity (≤ or > 20mitoses/50 HPF). The histological differential diagnosis be- tween ACA and ACC can be easy in some is straightforward in most cases. The former is characterized by nests and cords of lipid-rich and lipid-poor cortical cells arranged in varying proportion, showing low pleomorphism and absent or low mitotic activity. A separate type of lipid-poor adenoma is the oncocytoma, exclusively composed by large cells with dense eosinophilic cytoplasm. ACC generally show solid,
* Corresponding author.
E-mail address: eliaguadagno84@gmail.com (E. Guadagno).
trabecular or large nested growth pattern and they often display a thick fibrous capsule. Necrosis is present in high grade forms, as well as high mitotic activity, also atypical. Nuclei are more pleomorphic than in ACA. Venous, sinusoidal and capsular invasion can be easily evident in ACC cases. The identification of a single parameter has low sensitivity for ACC diagnosis, hence multifactorial diagnostic algorithms have been proposed. Weiss score [4,5] is the most widely accepted and consists on the evaluation of nine morphologic parameters (high nu- clear grade, mitotic rate > 5 mitoses/50HPF, atypical mitotic fig- ures, < 25 % of clear cells, diffuse architecture, tumor necrosis, ve- nous/sinusoidal/capsular invasion): when the presence of three or more of these parameters is suggestive of malignancy are present, the lesion is classified as malignant. Oncocytic lesions, instead, are categorized according to Lin-Weiss-Bisceglia criteria [6]: the presence of 1 major criteria (among mitotic rate < 5 mitoses/50HPF, atypical mitoses, ve- nous invasion) is indicative of malignancy, while the presence of 1-4 minor criteria (among size > 10 cm and/or weight > 200 g, necrosis, sinusoidal invasion, capsular invasion) indicates uncertain malignant potential.
Both these algorithms are based on morphologic parameters that can be observer- dependent. In some borderline cases a high degree of discordance is observed [7]. More objective parameters should be in- cluded in the algorithm for the differential diagnosis between ACA and ACC. Molecular investigations [3] have proved to be of little use in this regard but have shown some essential aspects concerning ACC sub- typing: for example, it was shown that p53 mutations are more frequent in children or that WNT pathway deficits are present in 50 % of cases.
The adrenal medulla is composed of specialized neural crest (neu- roendocrine) cells (chromaffin cells) and their supporting cells (sus- tentacular cells). It is the major source of catecholamines (nor- epinephrine and epinephrine) in the body and Pheochromocytoma (tumor of the chromaffin cells) (PH) is the most important disease that can involve the adrenal medulla this district. Its incidence is around 0.4-9.5 cases/1 million population [8] and in 61 % of cases it is an incidental finding. In 10 % of cases they occur within clinical syn- dromes (eg. MEN2A, von Hippel Lindau, etc). Microscopically, it is characterized by nests of polygonal cells separated by peripheral ca- pillaries. Neoplastic cells can closely resemble normal chromaffin cells with basophilic to amphophilic cytoplasm. Nuclear pleomorphism can be conspicuous and mitoses are usually rare. Less commonly, cells may show a spindle shape or a clear cytoplasm (so-called lipid degenera- tion).
In the past, there was a general belief that it is almost always a benign neoplasm and it was called malignant only after the evidence of metastasis (10 % of cases). Currently, all phaeochromocytomas are believed to have some metastatic potential [8], hence a new approach, based on risk stratification, has been introduced. A prognostic role has been identified for five morphologic features, as invasion (vascular, adrenal capsular and periadrenal soft tissue), architectural variation (irregular, enlarged and confluent cell nests; diffuse growth), cytolo- gical variation (spindling, small cells, high cell density, cellular monotony, and pleomorphism), necrosis and proliferative activity (mitotic activity, atypical mitoses or Ki67). Controversial is the pre- dictive value of each individual factor [9,10]. The weakness of these potential prognostic factors stands in the extreme subjectivity of their evaluation, resulting in poor agreement between pathologists. Multi- factorial scoring systems (Table 1) have been produced (GAPP and PASS score) [11,10], in order to make a post-surgical risk stratification. Recently, it was shown [12] that these algorithms have a high negative predictive value, so they are excellent in “ruling out” rather than “ruling in” malignant potential in these neoplasms. Recently [8], the study of immunohistochemical expression was introduced as a surro- gate of the genetic testing to identify familial forms that are more likely to recur due to SDH gene mutations resulting in a loss of expression of the SDHB protein.
Aim of our study was to analyze the immunological cellular
| PASS SCORE | |
|---|---|
| Features | Score |
| Central tumor necrosis (at the center of large nests) or confluent tumor necrosis (not degenerative alterations) | 2 |
| High cellularity | 2 |
| Cellular monotony | 2 |
| Spindle cells (also focal) | 2 |
| > 3/10 HPF mitotic figures | 2 |
| Atypical mitotic figures | 2 |
| Fat tissue extension | 2 |
| Vascular invasion | 1 |
| Capsular invasion | 1 |
| High nuclear pleomorphism | 1 |
| Nuclear hyperchromasia | 1 |
| Total maximum score | 20 |
GAPP SCORE
| Features | Score | |
|---|---|---|
| Histologic pattern | Zellballen | 0 |
| Large and irregular nests | 1 | |
| Pseudorosettes (also focal) | 1 | |
| Cellularity | Low (< 150 cells/U) | 0 |
| Moderate (150-250 cells/U) | 1 | |
| High (> 250 cells/U) | 2 | |
| Comedonecrosis | Absent | 0 |
| Present | 2 | |
| Vascular or capsular invasion | Absent | 0 |
| Present | 1 | |
| Ki67 L.I. | < 1 % | 0 |
| 1-3 % | 1 | |
| > 3 % | 2 | |
| Catecholamines | Epinephrine | 0 |
| Norepinephrine | 1 | |
| Non-functioning | 0 | |
| Total maximum score | 10 | |
population (type, density and location of lymphocytes within the tumor) in these forms of mostly benign neoplasms. Tumor-infiltrating immune cells have been characterized in other malignant tumors [13-15], showing to be a better predictor of patient survival than other histopathological parameters. A few studied have been conducted on the prognostic role of inflammation in tissue derived from adrenocor- tical neoplasms. In a cohort of 48 primary ACC from children [16] it was observed that higher counts of CD8+-T cells were associated with a younger age and stage I. Much less is known regarding inflammatory conditions of PH.
Furthermore, PD-L1 immunohistochemical expression was assessed in ACC and in PH cases. A literature search showed that PD-L1 ex- pression was found in a minority of ACC cases [17] and that a high PD- L1 mRNA expression, analyzed on a cohort of 146 ACC, was associated with longer, post-operative survival [18]. By evaluation of gene-ex- pression profile in “high PD-L1 expressor” tumors, it was observed the existence of a signature whose ontology analysis was indicative of a cytotoxic T cells response.
Aim of our study was to analyze the immunological cellular popu- lation (type, density and location of lymphocytes within the tumor) in these forms of mostly benign neoplasms.
2. Methods
2.1. Collective
The collective included 15 cases of adrenocortical neoplasms (ADA and ACC), diagnosed between 2013 and 2019 and 17 cases of
pheochromocytomas (PH), diagnosed between 2013 and 2018. All cases were retrieved from the archives of the Institute of Pathology of Federico II University Hospital of Naples. All the cases were classified according to the 2017 World Health Organization Classification system [1,3,8] and staged (ACC and PH) according to the American Joint Committee on Cancer (AJCC VIII edition) System [19,20]. No syn- dromic forms were present.
The slides were reviewed by two experienced pathologists (MD, EG), in order to define, more precisely, the diagnosis of ACC and ACA, through Weiss criteria and the modified Lin-Weiss-Bisceglia version, for oncocytic lesions. Phaeochromocytomas were examined again at light microscope to identify prognostic parameters for PASS and GAPP scores quantification.
All the specimens were formalin-fixed and paraffin-embedded. The most significant paraffin block was selected and from each block three (four) 4 pm-thick sections were cut to perform immunostaining with antibodies anti CD45 (marker of hematopoietic cells except ery- throcytes and platelets), anti-CD20 (B cell marker) and anti-CD3 (T cell marker) and PD-L1.
A signed informed consent to use surgical specimens for scientific purposes was available for each patient.
2.2. Immunohistochemistry
Sections were dewaxed in xylene, hydrated in graded series of al- cohol and subjected to heat-induced antigen retrieval (10 mM Sodium Citrate, 0.05 % Tween 20, pH 6.0). After blocking endogenous perox- idase activity, the tissue was incubated with monoclonal antibodies for CD45 (EP322Y clone, rabbit, Ventana, 1:100 dilution) or CD20 (L26 clone, mouse, Ventana, 1:100 dilution) or CD3 (2GV6, rabbit, Ventana, 1:100 dilution), all for 90 min. Subsequently, the slices were rinsed and incubated with the biotinylated secondary antibody, at room tem- perature, for 30 min. The bound antibody complexes were stained for 3-5 min or until appropriate for microscopic examination with diami- nobenzidine, and they were then counterstained with hematoxylin (30 s) and mounted. For all the cases Ki67 (30-9 clone Ventana) im- munostaining had been performed according to automated procedure. PD-L1 (SP263 clone, Ventana) was automatically assessed in 4 ACC and in 17 PH cases, Beta-catenin (14 clone, mouse, Ventana) and p53 (D07 clone, mouse, DAKO) were assessed in the 4 ACC cases.
2.3. Immunoscore
For CD45, CD20 and CD3 staining evaluation a manual count was performed. 16 regions were examined, each measuring 1 mm2 and, of these, 8 were located at the periphery (P, within 3 mm deep from the capsule) and 8 were central (C, over half the diameter of the lesion) (Fig. 1a and a). The 16 examined regions were the same in the 3 markers. Evaluations were made by two independent observers and, in discordant cases, within 5 % of the value, the slides were reviewed at multi-head microscope to achieve consensus.
The signal was expressed as nr of cells/ mm2 and, for each case, a median value was calculated at the periphery of the tumor (P), a median value at the center (C) and, finally, a total median (T) was provided from the two other values (P and C).
Ultimately, the inflammatory lymphocytic infiltrate was character- ized for cell type, its density and its location within the tumor.
For the evaluation of Ki67 Labeling Index, “hot spot” areas were chosen at low magnification and an average of the values obtained on 5 adjacent fields (at least 500 neoplastic cells) was calculated [21]. When the score was discordant, it was assessed again, collegially, by manually counting unlabeled and labeled nuclei on a camera-captured, printed image.
2.4. Statistical evaluation
In order to compare the (T) scores obtained with the three markers, ANOVA variance analysis was applied. t-test was used for comparison between P and C score for each marker.
The association between markers and clinico-pathologic variables was studied by Fisher’s exact test.
A p value ≤ 0.05 was considered statistically significant. All tests were two sided and carried out with GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA).
3. Results
3.1. Clinico-pathologic features in ACA and ACC patients
Of 15 (Table 2) patients, 9 were females and 6 were males, aged between 35 and 74 years (with a mean age of 55 years and a median age of 57 years). The lesion originated from the right adrenal gland in 10 cases and from the left adrenal gland in 5 cases. Its maximum dia- meter ranged from 1 to 24 cm (mean value: 6 cm; median value: 5 cm). Tumor weight ranged from 8 to 2600 g, with a mean value of 120 g and a median of 120 g.
After slides revision, it was observed a diffuse architecture in 8 cases, necrosis in 4 cases, high nuclear grade in 4 cases, clear cell portion in < 25 % of the tumor in 7 cases, capsular invasion and venous invasion in 2 cases. Mitotic activity was never > 5 mitoses/50HPF and in one case atypical mitoses were evident.
By applying the Weiss criteria and Lin-Weiss-Bisceglia criteria for oncocytic lesions, the diagnosis was ACA in 10 cases, oncocytic lesion with uncertain malignant potential (OLUMP) in 1 case that had pre- viously been diagnosed as ACC, and ACC in 4 cases.
The reclassification of ACC to OLUMP was possible because two Weiss criteria (diffuse growth pattern and clear cell portion < 25 %) that are implicit features of oncocytic lesions, were not considered. Only necrosis remained which, however, was a minor criterion and not sufficient to define this lesion as malignant.
Ki67 Labeling Index was calculated in all cases and showed a value that ranged from 2 to 10 %, with a mean value of 4 % and a median of 3 %.
All ACC cases were negative to PDL1, Beta-catenin and p53.
3.2. Immunoscore in ACA and ACC patients
CD45+ cells showed a (T) mean density of 32 cells/mm2 (median value of 23 cells/mm2), with a mean (P) value of 32 cells/mm2 (median value of 19 cells/mm2) and of 33 cells/mm2 (median value of 22 cells/ mm2) in the central zone (C). CD20+ cells showed a (T) mean density of 6 cells/mm2 (median value of 0 cells/mm2), with a mean (P) value of 7 cells/mm2 (median value of 0 cells/mm2) and of 4 cells/mm2 (median value of 0 cells/mm2) in the central zone (C). CD3 + cells showed a (T) mean density of 42 cells/mm2 (median value of 27 cells/mm2), with a mean (P) value of 37 cells/mm2 (median value of 22 cells/mm2) and of 43 cells/mm2 (median value of 32 cells/mm2) in the central zone (C).
The difference of cellular density between the three markers was statistically significant (p value = 0.0028), finding the highest values for CD45 and CD3, compared to CD20 (Fig. 1b). Therefore, the in- flammatory infiltrate was mainly attributable to T cells.
Furthermore, it was shown that no differences in distribution of cellular density between the periphery (P) and the center (C) of the tumor were evident (Fig. 1c).
3.3. Correlation between Immunoscore and clinico-pathological features in ACA and ACC patiens
Cases were divided into two groups, using a 10 cells/mm2 cut-off, to perform correlation studies with clinico-pathological features. Since no
p= 0.0028
150-
CD45
CD3
cells/ mm2
CD20
100-
50
TAT
0
Inflammatory cells
B cells
T cells
b
periphery (P) center (C)
1mm2
200-
CD45
CD3
o
cells/ mm2
150-
CD20
o
100-
50
0
Inflamm cells
B cells
T cells
a
C
| Parameter | Measures |
|---|---|
| Age (years) | mean: 55, median: 57, range: 35-74 |
| Sex | males: 6, females: 9 |
| Site | right: 10, left: 5 |
| Maximum diameter (cm) | mean:6, median: 5, range: 1-24 |
| Weight (g) | mean: 120, median: 120, range: 8-2600 |
| Diagnosis | adenoma: 10; OLUMP *: 1; carcinoma: 4 |
| Diffuse architecture | 8 cases |
| Necrosis | 4 cases |
| High nuclear grade | 4 cases |
| > 5 mitoses/50HPF | 0 |
| Atypical mitoses | 1 case |
| < 25 % clear cell | 7 cases |
| Capsular invasion | 5 cases |
| Vascular invasion | 2 cases |
| Ki67 (%) | mean: 4, median: 3; range: 2-10 |
* OLUMP: oncocytic lesion with uncertain malignant potential.
difference was detected between (P) and (C) immunoscores (Table 3A), (T) immunoscore was preferred for these studies. Fisher’s exact test (Table 4) revealed a statistically significant (p = 0.033) association between high CD45+ and CD3+ cellular density and ACA diagnosis (Fig. 3). The case that was reclassified as OLUMP (case 12) showed a high total CD45+ and CD3 + cellular density (both > 10 cells/mm2).
Furthermore, although not statistically significant, there was a tendency to observe a higher (T) immunoscore in lesions with a lower weight (p value = 0.077) and in those with Ki67 L.I. < 5 % (p value = 0.077). No correlation was found with any clinico-pathological variables included in Weiss score (Supplementary Table 1), but a slight tendency towards statistical significance between high CD45 density (≥10cells/mm2) and the absence of vascular invasion (p value = 0.0769).
3.4. Clinico-pathologic features in PH patients
Of 17 cases (Table 5), 5 were males and 12 females, aged between 29 and 78 years (mean age of 48 years, median age of 47 years). The lesion originated from the right adrenal gland in 7 cases and from the
left adrenal gland in 8 cases. In 1 case the metastatic site was examined. The maximum diameter measured between 2.5 and 10.5 cm (mean value of 7.4 cm, median of 8 cm) and the pathological staging found was pT1Mx (< 5 cm) in 4 cases, pT2Mx (≥5 cm) in 11 cases, pT3Mx (with evidence of fat tissue extension) in 1 case and pT3M1 in 1 case (metastasis of the anterior abdominal wall). Tumor weight ranged from 10 to 145.4 g, with a mean value of 64 g and a median of 58 g. After the revision of Haematoxylin and eosin-stained slides, it was observed a diffuse growth pattern in > 10 % of the neoplasm in 10 cases, the presence of necrosis in 4 cases, hypercellularity in 7 cases, cellular monotony in 7 cases, cellular “spindling” in 5 cases, mitotic activity > 3 mitoses/10 HPF in 2 cases, also with atypical mitoses. Furthermore, the lesion invaded perivisceral fat tissue in 2 cases and showed capsular and vascular invasion, respectively in 3 and 6 cases. Cells were pleo- morphic in 10 cases and nuclei hyperchromatic in 8 cases. Ki67 La- beling Index was < 1 % in 9 cases, between 1 and 3 % in 3 cases and > 3 % in 5 cases. Respectively in 2 and 6 cases norepinephrine and epinephrine was secreted, while in 9 cases the neoplasm was non- functioning. Consequently, PASS score ranged from 2 to 12 and GAPP score from 0 to 5 (Fig. 4). In the metastatic case, both PASS and GAPP score had the highest value (respectively 12 and 5). All PH cases were negative to PD-L1.
3.5. Immunoscore in PH patients
CD45+ cells showed a (T) mean density of 17 cells/mm2 (median value of 11 cells/mm2), with a mean (P) value of 17 cells/mm2 (median value of 12 cells/mm2) and of 16 cells/mm2 (median value of 12 cells/ mm2) in the central zone (C). CD20+ cells showed a (T) mean density of 4 cells/mm2 (median value of 0 cells/mm2), with a mean (P) value of 5 cells/mm2 (median value of 0 cells/mm2) and of 2 cells/mm2 (median value of 0 cells/mm2) in the central zone (C). CD3 + cells showed a (T) mean density of 18 cells/mm2 (median value of 16 cells/mm2), with a mean (P) value of 20 cells/mm2 (median value of 15 cells/mm2) and of 15 cells/mm2 (median value of 14 cells/mm2) in the central zone (C) (Table 3B).
A clear difference of cellular density between the three markers was evident and it was statistically significant (p value = 0.0049), with higher values for CD45 and CD3, compared to CD20 (Fig. 2b).
| A | Adrenocortical neoplasms | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Cases | CD45(T) cells/mm2 2 | CD45(P) cells/mm2 2 | CD45(C) cells/mm2 2 | CD20(T) cells/mm2 2 | CD20(P) cells/mm2 2 | CD20(C) cells/mm2 2 | CD3(T) cells/mm2 2 | CD3(P) cells/mm2 2 | CD3(C) cells/mm2 2 |
| 1 | 61 | 70 | 52 | 19 | 38 | 0 | 128 | 85 | 171 |
| 2 | 73 | 81 | 65 | 28 | 36 | 21 | 87 | 96 | 78 |
| 3 | 38 | 69 | 8 | 5 | 10 | 0 | 38 | 64 | 12 |
| 4 | 13 | 14 | 13 | 0 | 0 | 0 | 27 | 22 | 32 |
| 5 | 18 | 19 | 18 | 0 | 0 | 0 | 8 | 9 | 7 |
| 6 | 30 | 26 | 35 | 0 | 0 | 0 | 42 | 28 | 57 |
| 7 | 62 | 34 | 91 | 9 | 3 | 16 | 79 | 20 | 138 |
| 8 | 3 | 5 | 1 | 0 | 0 | 0 | 2 | 0 | 4 |
| 9 | 17 | 10 | 25 | 0 | 0 | 0 | 27 | 30 | 25 |
| 10 | 23 | 19 | 27 | 0 | 0 | 0 | 27 | 20 | 34 |
| 11 | 9 | 5 | 14 | 0 | 0 | 0 | 11 | 6 | 16 |
| 12 | 34 | 46 | 22 | 0 | 0 | 0 | 47 | 54 | 40 |
| 13 | 91 | 54 | 128 | 19 | 15 | 23 | 93 | 111 | 75 |
| 14 | 3 | 4 | 1 | 0 | 0 | 0 | 7 | 12 | 2 |
| 15 | 9 | 17 | 1 | 3 | 6 | 1 | 1 | 2 | 0 |
| B | Phaeochromocytomas | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Cases | CD45(T) cells/mm2 2 | CD45(P) cells/mm2 2 | CD45(C) cells/mm2 2 | CD20(T) cells/mm2 2 | CD20(P) cells/mm2 2 | CD20(C) cells/mm2 2 | CD3(T) cells/mm2 2 | CD3(P) cells/mm2 | CD3(C) cells/mm2 2 |
| 1 | 49 | 73 | 25 | 9 | 16 | 2 | 47 | 86 | 8 |
| 2 | 17 | 21 | 13 | 0 | 0 | 0 | 9 | 4 | 14 |
| 3 | 27 | 24 | 30 | 8 | 10 | 5 | 20 | 19 | 20 |
| 4 | 7 | 10 | 3 | 0 | 0 | 0 | 6 | 2 | 9 |
| 5 | 15 | 18 | 12 | 1 | 1 | 0 | 15 | 15 | 14 |
| 6 | 20 | 14 | 26 | 0 | 0 | 0 | 16 | 11 | 21 |
| 7 | 15 | 17 | 13 | 0 | 0 | 0 | 18 | 15 | 20 |
| 8 | 7 | 8 | 5 | 0 | 0 | 0 | 17 | 23 | 11 |
| 9 | 6 | 7 | 4 | 0 | 0 | 0 | 9 | 9 | 8 |
| 10 | 11 | 13 | 9 | 1 | 1 | 0 | 11 | 10 | 12 |
| 11 | 19 | 12 | 27 | 1 | 1 | 0 | 31 | 24 | 38 |
| 12 | 11 | 11 | 12 | 0 | 1 | 0 | 19 | 21 | 17 |
| 13 | 6 | 6 | 5 | 0 | 0 | 0 | 12 | 11 | 13 |
| 14 | 4 | 2 | 2 | 0 | 0 | 0 | 5 | 7 | 3 |
| 15* | 7 | 11 | 4 | 0 | 0 | 0 | 2 | 2 | 2 |
| 16 | 59 | 49 | 69 | 47 | 58 | 35 | 37 | 45 | 28 |
| 17 | 6 | 2 | 10 | 0 | 0 | 0 | 30 | 41 | 20 |
* This case was an abdominal metastasis from PH.
Therefore, the inflammatory infiltrate was mainly composed by T cells (Fig. 5). Furthermore, the immunoscore was homogeneous for all the three markers, both in the periphery (P) and in the center (C) of the lesion (Fig. 2c).
3.6. Correlation between Immunoscore and clinico-pathological features in PH patients
In PH patients, studies of association were conducted like in ACA and ACC patients.
Fisher’s exact test revealed just a higher (T) immunoscore in neo- plasms with weight > 60 g, however not statistically significant. No correlation was found with any clinico-pathological variables included in the GAPP and PASS scores (Supplementary Table 2).
The metastatic case showed a low immunoscore (CD45+ mean value: 7 cells/mm mm2, CD20+ mean value: 7 cells/mm mm2, CD3 + mean value: 2 cells/mm mm2).
4. Discussion
Aim of the present study was to define the type, density and dis- tribution within the tumor of the inflammatory cells in the adrenal neoplasms.
The main finding of the present study was that cells of neoplastic
inflammation were mainly T cells, with a 9:2 T cells/ B cells ratio. Their distribution appeared to be homogeneous, without specific gradient from the center to the periphery.
The role of T cells within the tissue is well known [22]. They are memory T cells that reside in the tissues and co-express CD3 and CD8. Their function is to form a barrier against the environment and to provide on-site defenses against pathogens. Their peculiarity stands in the ability to act faster than circulating memory T cells, because they already lie in the attack site. As “tissue explorers”, once they identify a foreign agent, they recruit other leucocytes and other cells to partici- pate to tissue defense. This is the normal immunological cell population of a non-neoplastic tissue.
On the base of what was described till know, a higher inflammatory cellular density was expected to be present at the periphery of the le- sion, under the capsule, in direct contact with the surrounding normal parenchyma. Instead, in our cases, the inflammatory cell density was homogeneous throughout the lesion.
It is known that, under neoplastic conditions, immunological de- fenses undergo alterations depending on the balance between pro-in- flammatory factors and immunosuppression.
In recent years, increasingly innovative data have been accumulated in the field of tumor immunology. In fact, it has been shown that the immune system is crucial in controlling tumor progression and that one of the major biological markers of tumor development is the ability to
| Variables | CD45 (T) | CD20 (T) | CD3 (T) | ||||||
|---|---|---|---|---|---|---|---|---|---|
| < 10cells/mm2 | ≥10cells/mm2 | p value | < 10cells/mm2 | ≥10cells/mm2 | p value | < 10cells/mm2 | ≥10cells/mm2 | p value | |
| Age | |||||||||
| < 55 ys | 1 | 5 | 0.604 | 4 | 2 | 0.5253 | 1 | 5 | 0.604 |
| ≥55 ys | 3 | 6 | 8 | 1 | 3 | 6 | |||
| Sex | |||||||||
| M | 2 | 7 | 1.000 | 7 | 2 | 1.000 | 3 | 6 | 0.604 |
| F | 2 | 4 | 5 | 1 | 1 | 5 | |||
| Weigth | |||||||||
| < 100g | 1 | 9 | 0.077 | 7 | 3 | 0.505 | 1 | 9 | 0.077 |
| ≥100g | 3 | 2 | 5 | 0 | 3 | 2 | |||
| Size | |||||||||
| < 5 cm | 1 | 7 | 0.282 | 5 | 3 | 0.200 | 1 | 7 | 0.2821 |
| ≥5 cm | 3 | 4 | 7 | 0 | 3 | 4 | |||
| Diagnosis* | |||||||||
| Adenoma | 1 | 10 | 0.033 | 8 | 3 | 0.516 | 1 | 10 | 0.033 |
| Carcinoma | 3 | 1 | 4 | 0 | 3 | 1 | |||
| Weiss | |||||||||
| < 3 | 1 | 8 | 0.235 | 6 | 3 | 0.229 | 1 | 8 | 0.235 |
| ≥3 | 3 | 3 | 6 | 0 | 3 | 3 | |||
| Ki67 | |||||||||
| < 5 % | 1 | 9 | 0.077 | 7 | 3 | 0.505 | 1 | 9 | 0.077 |
| ≥5 % | 3 | 2 | 5 | 0 | 3 | 2 | |||
* OLUMP was included among adenomas cases.
| Parameter | Measures |
|---|---|
| Age (years) | mean: 48, median: 47, range: 29-78 |
| Sex | males: 5, females: 12 |
| Site | right: 7, left: 9, metastasis: 1 |
| Maximum diameter (cm) | mean:7.4, median: 8, range: 2.5-10.5 |
| Weight (g) | mean: 64, median: 58, range: 10-145.4 |
| Pathological stage (AJCC VIII ed) | pT1Mx: 4, pT2Mx: 11,pT3 Mx:1; pT3M1:1 |
| Diffuse growth pattern > 10 % | score 0: 7 cases, score 2: 10 cases |
| Necrosis | score 0: 13 cases, score 2: 4 cases |
| Hypercellularity | score 0: 11 cases, score 2: 6 cases |
| Cellular monotony | score 0: 10 cases, score 2: 7 cases |
| "Spindling" | score 0: 12 cases, score 2: 5 cases |
| > 3mitoses/10HPF | score 0: 15 cases, score 2: 2 cases |
| Atypical mitoses | score 0: 15 cases, score 2: 2 cases |
| Fat tissue extension | score 0: 15 cases, score 2: 2 cases |
| Capsular invasion | score 0: 14 cases, score 1: 3 cases |
| Vascular invasion | score 0: 11 cases, score 1: 6 cases |
| Cellular pleomorphism | score 0: 7 cases, score 1:10 cases |
| Nuclear hyperchromasia | score 0: 9 cases, score 1: 8 cases |
| Ki67 | score0: 8 cases, score1: 3 cases, score 2: 6 cases |
| Catecholamine | score 0: 15 cases, score1: 2 cases |
escape immunosurveillance. For example, in colorectal cancer [23], the type, density and location of inflammation lymphocytic infiltrate have a prognostic significance. Immunoscore, in this area, would seem to have an even greater role than pathological TNM staging, in making a correct prognostic stratification. A greater inflammatory response of lympho- cytes would be associated with a better prognosis.
The inflammatory response is part of the microenvironment [24] which, nowadays, is being widely studied in several cancer types, in order to select the best therapeutic approach. The microenvironment includes all non-cancerous cells (fibroblasts, immune cells, blood ves- sels) or proteins (growth factors, interleukins) within the tumor, that may affect tumor behavior and response to therapies.
Among our collective of adrenocortical neoplasms, considerably higher immunoscore was recorded in benign, compared to malignant forms. This could support a possible immunosuppressive role of tumor cells in malignant transformation. In one case, the inflammation as- sessment supported the re-classification of an ACC case to OLUMP: the
lesion was characterized by a high density- inflammatory lymphocytic infiltrate, just like it was never observed in malignant cases. It could be interesting to evaluate any eventual differences in inflammatory cells lymphocytes density among ACC in a larger collective. More than 20 years ago [25], studies on cell cultures showed an immune escape of ACC because of an altered Fas/Fas-L system expression and loss of MHC class H expression.
However, a potential prognostic and diagnostic role could be re- cognized to lymphocytic inflammatory infiltrate in adrenocortical neoplasms.
They are endocrine lesions, sometimes with secretory activity that could, in some way, influence the inflammation through release of circulating substances or by paracrine action. Endocrine factors are relevant for tumor microenvironment [26].
Less explored was the knowledge of inflammation in tissues derived from PH. A Pubmed search was made by combining the following words: phaeochromocytoma/pheochromocytoma AND inflammation OR lymphocytes OR CD3 OR CD8 OR CD20 OR PDL1/PDL-1. No spe- cific papers were found out.
In our PH cases, in most of them the immunoscore was high and no differences were recorded between cases with different prognostic risk (based on GAPP and PASS score). The lesion behaved just like benign adrenocortical neoplasms. Interestingly, however, the only metastatic case showed a low immunoscore. This finding could furtherly support a role of immunosuppression in neoplastic malignant progression of adrenal lesions.
Most of literature data concern the study of inflammation in clearly malignant tumors. PH are tumors considered to be all potentially ma- lignant, for which prognostic scores are finalized in making a risk stratification. It could be useful to add inflammation as additional morphologic parameter in the prognostic algorithm, because it is less subjective than others. But more insights into this issue are needed, by analyzing a wider cohort of metastatic cases.
Another finding of the present study was the poor presence of B cells in tissues, as it was expected. Indeed, in physiological condition, B lymphocytes play a fundamental role in regulating humoral immunity through the production of immunoglobulins, but they are scarcely re- presented in tissues. Recently, additional functions have been demon- strated [27] for these cells, such as the antigen-presentation ability, the production of multiple cytokines and a suppressive action, mainly
p=0.0049
CD45
CD3
80-
CD20
cells/ mm2
60
40-
20-
0
Inflammatory cells
B cells
T cells
b
periphery (P)
1mm2
center (C)
100-
CD45
CD3
cells/ mm2
80-
CD20
60-
40-
20-
0
a
Inflamm. cells
B cells
T cells
C
ascribed to the production of IL-10. Convincing data have shown that these cells (Breg) can suppress inflammatory responses in experimental encephalomyelitis, collagen-induced arthritis and colitis. In our cases, this immunosuppressive action has not been shown.
As in other fields, the use of immunoscore, to quantify the intra- tumor inflammatory lymphocytic infiltrate, has important implications in clinical practice, for therapeutic management. At the basis of the immunological escape mechanism implemented by neoplastic cells would be the so-called “exhausted” T cells which overexpress multiple inhibitory receptors, including PD1 (programmed cell death 1), CTLA4 (cytotoxic T-lymphocyte antigen 4) and others [28,29]. The most re- levant finding is that antibodies directed against and that block the
inhibitory molecules of this checkpoint have proved useful in the treatment of some solid tumors (melanoma, non-small cell lung cancer, etc.). The actual clinical response has shown how it is possible to sti- mulate the function of anti-tumor endogenous T cells. Unfortunately, not all patients have shown the same type of response for which it is essential to identify predictive markers of therapy efficacy.
In our cases no expression of PDL1 clone was detected, so no pre- dictive role could be recognized. In this field, the immunotherapy does not seem to be very promising, indeed the phase I JAVELIN study failed to demonstrate a substantial activity of the PD-L1 inhibitor Avelumab in advanced ACC [30].
Overall, to the best of our knowledge, this is the first study assessing
a
b
&
0
d
PASS
score 1
2
score 3
1
score 4
1
score 5
2
score 6
5
score 7
2
score 8
2
score 10
1
score 12
1
GAPP
score 0
1
score 1
1
score 2
6
score 3
3
score 4
4
score 5
2
a
b
the expression of lymphocytic markers in the neoplasms of the adrenal glands and whose main finding is the detection of a higher immuno- score in cases of ACA compared to ACC. In PH cases inflammation was
reduced only in the metastatic case. Therefore, inflammation could represent a valuable diagnostic and potential prognostic parameter, useful for the correct management of these lesions.
Compliance with ethical standards
All procedures performed in studies were in accordance with the ethical standards of the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Funding
The study was carried out within the network of the project RIS 3- RARE.PLAT.NET.
CRediT authorship contribution statement
Elia Guadagno: Conceptualization, Formal analysis, Writing - ori- ginal draft. Daniela Russo: Data curation, Formal analysis. Sara Pignatiello: Methodology, Formal analysis. Marialaura Del Basso De Caro: Writing - review & editing, Project administration, Conceptualization.
Declaration of Competing Interest
The authors declare that they have no conflict of interest.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.prp.2020.153070.
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