ORIGINAL ARTICLE
The Utrecht Score: A novel histopathological scoring system to assess the prognosis of dogs with cortisol-secreting adrenocortical tumours
Karin Sanders1 | Koen Cirkel2 | Guy C. M. Grinwis2 | Erik Teske1 | Sebastiaan A. van Nimwegen 1 Jan A. Mol1 İD Jan Willem Hesselink1
Hans S. Kooistra1 | Sara Galac1
1Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands
2Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands
Correspondence
Sara Galac, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 108, 3584 CM, Utrecht, The Netherlands. Email: s.galac@uu.nl
A cortisol-secreting adrenocortical tumour (ACT) is the cause of naturally occurring canine hypercortisolism in approximately 15% to 20% of cases. The differentiation between an adreno- cortical adenoma and carcinoma is usually based on histopathology. However, histopathological parameters have never been linked to the dogs’ survival. Moreover, in human medicine the inter-observer variability of some histopathological parameters that are used for ACTs is high. The objective of this study was to establish a reliable and easy-to-use histopathological scoring system for cortisol-secreting ACTs that can assess the prognosis of dogs after adrenalectomy. Cortisol-secreting ACTs of 50 dogs, collected between 2002 and 2015, were included in this study. Twenty histopathological features were assessed by one veterinary pathologist and one resident in veterinary pathology. In addition, the Ki67 proliferation index was assessed by two observers. Only parameters with intra- and inter-observer agreement scores (intra-class correla- tion or Cohen’s kappa coefficient) of ≥0.40 were included in survival analyses. Use of multivari- ate forward stepwise regression analysis with associated hazard ratios led us to a scoring system which we call the Utrecht score: the Ki67 proliferation index, +4 if more than 33% of the tumour cells have clear/vacuolated cytoplasm and + 3 if necrosis is present. Using cut-off values of 6 and 11, we could distinguish three groups that had significantly shorter survival times with increasing Utrecht scores. We conclude that the Utrecht score can be used to assess the prognosis of dogs with cortisol-secreting ACTs after adrenalectomy, which can help to select high-risk dogs that might benefit from adjuvant treatment or additional monitoring.
KEYWORDS
adrenocortical adenoma, adrenocortical carcinoma, Cushing syndrome, dogs, Ki-67 antigen, pathology
1 INTRODUCTION
Naturally occurring hypercortisolism is one of the most common endo- crine disorders in dogs. It is caused by a cortisol-secreting adrenocortical tumour (ACT) in approximately 15% to 20% of cases, for which the treat- ment of choice is adrenalectomy, if no metastases can be detected.1
Without the presence of metastases, assessing the malignancy of an ACT remains challenging. The differentiation between an adreno- cortical adenoma (ACA) and adrenocortical carcinoma (ACC) is usually based on histopathology. Although several studies have been con- ducted on the histopathological analysis of human ACTs,2-9 the litera- ture on canine ACTs is less extensive. The most recent study on
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. @ 2019 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.
histopathology of canine ACTs was published in 2004, in which the authors compared a number of histopathological criteria between ACAs and ACCs.10 The criteria that were determined to be diagnosti- cally useful included tumour size, peripheral fibrosis, capsular invasion, trabecular growth pattern, haemorrhage, necrosis, single-cell necrosis, haematopoiesis, fibrin thrombi, cytoplasmic vacuolation and the prolif- eration index by use of the Ki67 marker.10 However, whether the presence or absence of these parameters was associated with poor survival times has not been assessed. Moreover, the parameters used in this study were analysed by only one pathologist,10 whereas in the classification of human ACTs the inter-observer variability is known to be high for some of these parameters.2,3
In humans, the most widely used histopathological scoring system to differentiate ACCs from ACAs is the Weiss score. In this system, the presence of three or more out of nine established histopathologic criteria indicates malignant potential.6,7 Although the Weiss score can diagnose an ACC with high sensitivity and specificity, some of the criteria included suffer from inter-observer variation and require eval- uation by an experienced endocrine pathologist.2,3 To reduce this inter-observer variation, other scoring systems such as the Weiss revisited score3 and the Helsinki score2 have been proposed. In addi- tion, several studies have shown the value of immunohistochemical staining for the proliferation marker Ki67 to differentiate between ACAs and ACCs.3,11-14
In humans, the prognosis of patients with an ACC does not only differ from that of patients with an ACA, but the prognosis also varies greatly within the group of patients with an ACC.8,15 In human ACCs, histopathological criteria that are associated with a poor prognosis include high mitotic rate, high Ki67 proliferation index (PI), and high Helsinki score.7,8,16,17
The objective of this retrospective study was to establish a reli- able and easy-to-use histopathological scoring system for cortisol- secreting ACTs that can assess the prognosis of dogs after adrenalectomy.
2 MATERIALS AND METHODS
2.1 Case selection
Canine cortisol-secreting ACTs were collected between 2002 and 2015. Permission to use the ACT tissue was obtained from all dog owners. The suspicion of hypercortisolism was based on the presence of clinical signs and routine laboratory findings consistent with hyper- cortisolism. Non-suppressible hypercortisolism was diagnosed using the low-dose dexamethasone suppression test, or urinary corticoid: creatinine ratios (UCCRs) combined with a high-dose dexamethasone suppression test.18 The presence of an adrenal tumour was visualized by abdominal ultrasonography, computed tomography, or both. All dogs underwent unilateral adrenalectomy, which was performed by one of four experienced veterinary surgeons. Dogs were excluded from the study when they were euthanized or died before, during, or within 2 weeks after adrenalectomy; when the dog had bilateral adre- nal tumours; when metastases were detected before or at time of
surgery; when no formalin-fixed paraffin-embedded tissue was avail- able; and when less than 3 months of follow-up information was available.
2.2 Clinical parameters
The dogs’ medical records were retrospectively reviewed for dog- related parameters, clinical tumour-related parameters and surgery- related parameters. Dog-related parameters included sex and neutering status, body weight, age at time of surgery and preoperative treatment for hypercortisolism. Tumour-related parameters included the location of the tumour (left or right), whether there was evidence of venous invasion seen during diagnostic imaging or during surgery, and the tumour diameter (not including normal adrenal tissue). Mea- surement of the tumour diameter was performed during diagnostic imaging (ultrasound or CT) and/or after surgery. Surgery-related parameters included the duration of the surgery, and whether the tumour capsule ruptured during surgery.
2.3 Histopathological parameters
The ACTs were collected within 10 minutes after surgical removal. A representative section of the tumour was fixed in formaldehyde for histopathology and immunohistochemistry, the remaining tumour sec- tions were used for other research purposes. The tissues were fixed in 4% buffered formaldehyde for 24 to 48 hours, embedded in paraffin, and cut into 4-um sections. One tissue section for each ACT was sta- ined with haematoxylin and eosin, and one with the Gordon and Sweet’s reticulin stain. Twenty histopathological parameters were assessed by one veterinary pathologist (G.C.M.G.) and one resident in veterinary pathology (K.C.): (a) reticulin fibre density; (b) growth pat- tern; (c) morphology of cytoplasm; the presence or absence of (d) haemorrhage, (e) extramedullary haematopoiesis, (f) intra-tumoural fibrosis, (g) peripheral fibrosis, (h) capsular invasion, (i) venous inva- sion, (j) sinusoidal invasion, (k) fibrin thrombi, (l) necrosis, (m) atypical mitotic figures, (n) nucleoli and (o) nuclear chromasia; and establish- ment of the (p) number of cells undergoing single-cell necrosis, (q) number of mitotic figures, (r) nuclear size, (s) cellular size, and (t) nuclear grade.
The reticulin fibre density was scored as the percentage of the ACT with decreased fibre density compared to the density of reticulin fibres in the zona fasciculata of a normal adrenal gland. The growth patterns were evaluated as being diffuse, nesting or trabecular, and the growth pattern that predominated was recorded. The reticulin fibre density and the growth patterns were evaluated in the Gordon and Sweet’s reticulin stain, all other parameters in the haematoxylin and eosin stain. An estimate of the percentage of cells with clear/vacuolated cytoplasm was noted. Peripheral fibrosis was consid- ered to be present when a multi-layered band of fibrous tissue sur- rounded at least part of the ACT. Capsular invasion was considered to be present when the ACT infiltrated or perforated the peripheral cap- sule; small nests of adrenocortical cells that resembled cells of the zona glomerulosa within the capsule were considered to be normal, because these nodules are also often encountered in adrenal glands of healthy dogs. Necrosis was considered to be present when
confluent nests of necrotic cells were visible. For the evaluation of single-cell necrosis and the number of mitotic figures, first the sec- tions were screened to determine where most cells undergoing single- cell necrosis or mitosis appeared to be present, and then these param- eters were evaluated in the one high power field (HPF; 400x magnifi- cation) where they seemed to be most abundant. Nuclear and cellular sizes were evaluated and given a number relative to a nucleus or cell in the zona fasciculata of a normal adrenal gland (eg, equally large: 1; twice as large: 2), the size that predominated was recorded. The nuclear grade was evaluated as described by Fuhrman et al. (1982).19 All histopathological parameters were visually estimated in the entire tissue section using a light microscope, no image analysis was per- formed. Both observers assessed the tissue sections twice at separate time points, and were blinded to the clinical data.
2.4 Ki67 proliferation index
For Ki67 immunohistochemistry, one tissue section per ACT was rehydrated in a series of xylene and ethanol baths. Antigen retrieval was performed with Tris-EDTA buffer (pH 9) in a microwave, at 850 W for 7 minutes and at 450 W for 15 minutes. After the slides were cooled down they were incubated with 0.35% H202 in Tris buff- ered saline (TBS) for 30 minutes to block endogenous peroxidase. The slides were blocked with 10% normal goat serum with 1% bovine serum albumin (BSA) in TBS for 30 minutes. Incubation with a mouse monoclonal primary anti-Ki67 antibody (MIB-1 clone, M7240, Dako, Agilent, Amsterlveen, The Netherlands), diluted 1:75 in 1% BSA in TBS, took place overnight at 4℃. The following day the slides were incubated with secondary antibody (HRP-labelled goat-anti-mouse, EnVision+, Dako) for 30 minutes. The slides were incubated with Dako Liquid DAB+ Substrate Chromogen System (K3468, Dako) for 10 minutes, and counterstained with haematoxylin. A series of etha- nol and xylene baths were used to dehydrate the slides, after which the slides were mounted with VectaMount Mounting Medium (H- 5000, Vector Laboratories, Peterborough, UK). During intermediate steps the slides were washed in TBS. Canine colon tissue slides were used as positive control tissue, and the primary antibody was replaced by normal mouse IgG (SC3877, Santa Cruz Biotechnology, Heidelberg, Germany) for the negative control.
The Ki67 PI was assessed by the resident in veterinary pathology (K.C.) and a researcher with experience in adrenocortical tumours (K.S.) in hot spot areas, which were the areas that appeared to have the highest percentage of Ki67 positive cells. For each tissue slide five images were captured on an Olympus BX60 microscope with Leica LAS-AF software, at 200x magnification. A minimum of 1000 nuclei were counted in total per ACT on the one or two images with the highest amount of Ki67 positive cells, for which Image software20 was used to keep track of the number of counted positive and nega- tive cells. Only nuclear staining was considered to be positive. Care was taken to only include ACT cells and not for example, cells of the stromal compartment or extramedullary haematopoiesis. The PI was calculated as the percentage of Ki67 positive nuclei relative to the total number of counted nuclei for each ACT.
2.5 | Analyses
The intra- and inter-observer agreement scores for histopathological parameters and the Ki67 PI were quantified using the intra-class cor- relation coefficient (ICCC) for continuous variables, and Cohen’s kappa coefficient for categorical variables. The strength of agreement was interpreted as follows: < 0.40, poor; 0.40-0.59, modere; 0.60-0.79, good; 0.80-1.00, excellent.21 Only parameters with an agreement score of more than 0.40 in all areas (ie, intra-observer agreement of both observers, and inter-observer agreement) were included in survival analyses. In case of continuous variables, the results of all four observations (ie, two observations per observer) were averaged for further analyses. In case of categorical variables, the result that was most prevalent was noted. If this could not be established, the parameter was scored again for these slides and the final outcome was noted.
Dogs were considered to have died as a result of the ACT when they were euthanized because of metastases or comorbidities related to recurrence of hypercortisolism. Recurrence of hypercortisolism was confirmed by the presence of hypercortisolism-related clinical signs and elevated UCCRs, which could be either because of regrowth of the ACT or metastases. For each dog, survival time was calculated from the time of surgery to euthanasia because of recurrence. If a dog died from unrelated causes, was lost to follow-up, or was still alive at the end of the study, then the dog was censored and the last known date that the dog was still alive was used as censoring date.
Univariate analyses were performed with the Cox proportional hazards model. All variables that had a P value of <0.15 in the univari- ate analyses were subsequently included in multivariate stepwise regression with forward selection. Optimal cut-off values were calcu- lated with receiver operating characteristic curves. The value with the highest Youden index (sensitivity + specificity - 1) was selected as the optimal cut-off value. Survival times were calculated using the Kaplan- Meier product-limit method. The log-rank method was used to calcu- late if differences between groups were significant.
P values of <0.05 were considered significant. All statistical ana- lyses were performed with SPSS Statistics for Windows (Version 24.0, IBM Corp, Armonk, New York).
3 RESULTS
3.1 Cases
A total of 50 dogs were included in the study. The most represented dog breeds were Labrador Retriever (5), Dachshund (5), Jack Russel Terrier (4), Schnauzer (2), Maltese (2), Fox Terrier (2) and White Shep- herd (2). Of the remaining dogs, 11 were mixed-breed dogs and 17 were of breeds that were represented once. Information on addi- tional dog-related parameters is included in Table 1.
The estimated median survival time after adrenalectomy for all 50 dogs as calculated by the Kaplan-Meier method was 54.7 months (95% CI 47.1-62.2 months) (Figure 1A). Of the 50 dogs, 19 were known to have had recurrence of hypercortisolism. The median sur- vival time of these 19 dogs with recurrence was 16.9 months (95% CI 10.8-49.3 months). All 31 dogs that had no recorded recurrence were
| Parameter | Distribution | Total | Hazard ratio (95%CI) | P value |
|---|---|---|---|---|
| Sex | 25 male, 25 female | 50 | 1.046 (0.420-2.607) | 0.921 |
| Neutered | 22 no, 28 yes | 50 | 1.483 (0.576-3.814) | 0.414 |
| Body weight | 14.2 (3.6-74.7) kg | 50 | 1.011 (0.979-1.045) | 0.506 |
| Age | 10.0 (2.1-12.9) y | 50 | 1.099 (0.844-1.431) | 0.483 |
| Treated before surgery | 36 no, 9 yes | 45 | 1.060 (0.346-3.250) | 0.918 |
| Location | 23 left, 25 right | 48 | 1.263 (0.502-3.176) | 0.620 |
| Venous invasion (macro) | 27 no, 16 yes | 43 | 0.722 (0.277-1.882) | 0.505 |
| Tumour diameter | 2.5 (1.0-10.0) cm | 46 | 1.424 (1.110-1.827) | 0.005* |
| Surgery duration | 156 (42-290) min | 43 | 1.002 (0.993-1.012) | 0.630 |
| Capsule rupture | 31 no, 16 yes | 47 | 1.339 (0.525-3.415) | 0.541 |
Univariate analyses performed with the Cox proportional hazards model. Distribution is indicated in categories for categorical variables, and in median (with the range in parentheses) for continuous variables. Total indicates the total number of dogs for which this parameter was known. Hazard ratio (with the 95% confidence interval in parentheses) indicates the hazard of the second category compared to the first in case of categorical parameters, and the hazard per stated unit in case of continuous variables. Significant P values are indicated in italic with an asterisk.
censored in the survival analyses so no estimated median survival was reached using the Kaplan-Meier method, but the median follow-up time was 27.1 months (95% CI 15.4-42.0 months).
3.2 | Clinical parameters
Of all clinical parameters that were analysed, only the tumour diameter was significantly associated with survival (univariate anal- ysis, Table 1). The optimal cut-off value was approximately 3 cm, which resulted in a significant (P = 0.034) difference in survival times between dogs with an ACT ≥3 cm (n = 18, estimated median survival time 24.2 months, 95% CI 1.3-47.1 months) and dogs with an ACT <3 cm (n = 28, estimated median survival time 54.7 months, 95% CI 48.2-61.1 months) (Figure 1B). Cut-off values of 2 or 5 cm as previously suggested in literature10,22 did not result in significant differences in survival times (P = 0.255 and P = 0.172, respectively).
3.3 Histopathological parameters and Ki67 PI
In assessing intra- and inter-observer reliability, four histopathological parameters had agreement scores of more than 0.4 in all areas (ie, intra-observer agreement scores for both observers, and inter- observer agreement score): decreased reticulin fibre density (Figure 2A, lowest agreement score 0.56, P < 0.001), extramedullary haematopoiesis (Figure 2B, lowest agreement score 0.59, P < 0.001), clear/vacuolated cytoplasm (Figure 2C and D, lowest agreement score 0.67, P < 0.001) and necrosis (Figure 2E, lowest agreement score 0.51, P < 0.001). No intra-observer agreement scores could be calcu- lated for the Ki67 PI (Figure 2F) because both observers assessed all ACTs once, but the inter-observer agreement score was excellent (0.96, P < 0.001). All intra- and inter-observer agreement scores are provided in Supporting Information Table S1.
In assessing the potential of the histopathological parameters as prog- nostic indicators, three parameters were significantly associated with sur- vival according to univariate analyses (Table 2): the percentage of
(A)
(B)
1.0
1.0
Cumulative survival
0.8
Cumulative survival
0.8
0.6
0.6
P = 0.034
0.4
0.4
3 cm
HR 2.81
0.2
0.2
≥ 3 cm
(1.08 - 7.30)
0.0
0.0
0
20
40
60
0
20
40
60
Survival (months)
Survival (months)
(A)
(B)
a
50 um
50 um
50 um
(C)
(D)
50 um
50 pm
(E)
(F)
100 um
50 um
| Parameter | Distribution | Total | Hazard ratio (95%CI) | P value |
|---|---|---|---|---|
| Decreased reticulin fibre density | 85 (19-100) % | 50 | 1.018 (0.987-1.051) | 0.257 |
| Clear/vacuolated cytoplasm | 48 (5-96) % | 50 | 1.023 (1.004-1.043) | 0.020* |
| Extramedullary haematopoiesis | 37 no, 13 yes | 50 | 0.882 (0.284-2.737) | 0.828 |
| Necrosis | 30 no, 20 yes | 50 | 3.495 (1.336-9.141) | 0.011* |
| Ki67 PI | 3 (0-22) % | 50 | 1.174 (1.065-1.295) | 0.001* |
Abbreviation: PI, proliferation index.
Univariate analyses performed with the Cox proportional hazards model. Distribution is indicated in categories for categorical variables, and in median (with the range in parentheses) for continuous variables. Total indicates the total number of dogs for which this parameter was known. Hazard ratio (with the 95% confidence interval in parentheses) indicates the hazard of the second category compared to the first in case of categorical parameters, and the hazard per stated unit in case of continuous variables. Significant P values are indicated in italic with an asterisk.
| Parameter | Distribution | Hazard ratio (95%CI) | P value |
|---|---|---|---|
| Ki67 PI | 3.1 (0.0-22.2) % | 1.124 (1.018-1.242) | 0.021* |
| Clear/vacuolated cytoplasm in ≥33% | 18 no, 32 yes | 4.350 (1.224-15.455) | 0.023* |
| Presence of necrosis | 30 no, 20 yes | 3.181 (1.103-9.173) | 0.032* |
Abbreviation: PI, proliferation index.
Multivariate stepwise regression performed with the Cox proportional haz- ards model with forward selection. Distribution is indicated in categories for categorical variables, and in median (with the range in parentheses) for con- tinuous variables. Hazard ratio (with the 95% confidence interval in paren- theses) indicates the hazard of the second category compared to the first in case of categorical parameters, and the hazard per stated unit in case of con- tinuous variables. Significant P values are indicated in italic with an asterisk.
clear/vacuolated cytoplasm, the presence of necrosis, and the Ki67 PI. For the continuous variables that were significantly associated with survival, we calculated optimal cut-off values, for which dogs that had recurrence and died within 30 months after surgery were included in the positive group (n = 13), and dogs that had no recorded recurrence and lived for at least 30 months were included in the negative group (n = 15). For the per- centage of clear/vacuolated cytoplasm, the optimal cut-off value was approximately 33%. For the Ki67 PI, the optimal cut-off value was 4.6%.
3.4 | The Utrecht score
To determine which parameters were independent predictors of poor survival, we used multivariate logistic regression with forward selection. This indicated that the Ki67 PI, clear/vacuolated cytoplasm in at least 33% of the tumour cells, and the presence of necrosis were all indepen- dent predictors of survival. Based on their hazard ratios in the multivari- ate analysis (Table 3), this led to the scoring system: 1.12 x Ki67 PI, + 4.35 if clear/vacuolated cytoplasm was present in at least 33% of the tumour cells, + 3.18 if necrosis was present. We simplified this system to: the Ki67 PI, + 4 if ≥33% of tumour cells have clear/vacuolated cyto- plasm, + 3 if necrosis is present, which we call the Utrecht score (Figure 3).
The median Utrecht score was 7.1 (range, 0.4-29.2), and the opti- mal cut-off value was approximately 6. To further divide the ACTs with scores of 6 or higher into two groups with different survival times, we used a cut-off value of 11, which was calculated as the approximate optimal cut-off value in the subset of patients with scores of more than 6. We then categorized the ACTs according to their Utrecht score in
three groups: (1) Utrecht score < 6 (n = 17), (2) Utrecht score ≥ 6 to <11 (n = 22) and (3) Utrecht score ≥ 11 (n=11). As illustrated in Figure 4, the estimated median survival time was not reached for group 1, was 51.5 months (95% CI 43.7-59.3) for group 2 (significantly differ- ent from group 1, P = 0.012), and 14.4 months (95% CI 10.5-18.2) for group 3 (significantly different from group 2, P = 0.005).
I
4 DISCUSSION
Here we introduce the Utrecht score: a novel histopathological scor- ing system that can assess the prognosis of dogs with a cortisol- secreting ACT after adrenalectomy. With cut-off values of 6 and 11, we could distinguish three groups that had significantly shorter survival times with increasing scores. We propose to classify ACTs with a score of <6 as having low risk of recurrence, with a score of ≥6 to <11 as having moderate risk of recurrence, and with a score of ≥11 as having high risk of recurrence.
Because most veterinary pathologists will not encounter an ACT on a daily basis, histopathological assessment of an ACT ideally should not require extensive training. In this study, 20 histopathological parameters were scored twice by both an experienced veterinary pathologist and a resident in veterinary pathology. This allowed us to include only those parameters that turned out to have low intra- and inter-observer variability irrespective of the observers’ experience, thereby improving the reliability of the Utrecht score.
A recent study on human ACCs reported low intra- and inter- observer agreement scores for Ki67 scoring among 14 trained endo- crine pathologists.23 However, in that study each observer was allowed to perform the analyses according to their own method of preference, which included visual estimation, formal manual count, and digital image analysis. In our study, the inter-observer agreement of the Ki67 PI was excellent, which is most likely associated with the methodology used. To standardize Ki67 PI scoring as much as possi- ble, we suggest to analyse the Ki67 PI in hot spot areas, to capture images of these areas, to count at least 1000 nuclei, and to use ImageJ20 or similar software to keep track of the number of counted positive and negative cells. A drawback of this method is that it is time-consuming, but developments in digital microscopy-enabled methods could facilitate future reproducible and reliable Ki67 PI assessments.23
50 pm
50 pm
100 pm
Ki67 proliferation index
+ 4
if ≥ 33% of cells have clear/vacuolated cytoplasm
+ 3 if necrosis is present
1.0
Cumulative survival
0.8
P = 0.012
< 6
HR 8.96
(1.15 - 69.75)
0.6
≥6,<11
P = 0.005
HR 4.73
≥ 11
(1.44 - 15.60)
0.4
0.2
0.0
0
20
40
60
Survival (months)
Increased cell proliferation and tumour hypoxia are both impor- tant features of aggressive cancers,24 which explains why Ki67 PI and necrosis are important prognostic factors in canine ACTs. Why the high percentage of clear/vacuolated cytoplasm is an important factor is less clear. The appearance of clear/vacuolated cytoplasm likely indi- cates the presence of intra-cytoplasmic lipid droplets, which are extracted during histologic specimen preparation unless special methods are used.25 Since cholesterol, which could be stored in these lipid droplets, functions as substrate for all steroid hormones, the per- centage of cells with clear/vacuolated cytoplasm could be related to the production of cortisol or its precursors. In human ACTs, cortisol production is known to be a negative prognostic indicator.26,27 In our study, however, only cortisol-secreting ACTs were included so the presence or absence of hypercortisolism was not a variable, but the percentage of cells with clear/vacuolated cytoplasm could have been
related to the degree of hypercortisolism. Because different methods or assays were used to establish hypercortisolism, we were unable to test this hypothesis. For future studies it would be interesting to determine whether clear/vacuolated cytoplasm is indeed related to the degree of cortisol production, since this would also indicate that non-secreting ACTs might be less malignant overall than cortisol- producing ACTs.
Of the clinical parameters, only the tumour diameter was signifi- cantly associated with survival. However, it did not retain its signifi- cance in the multivariate analysis, neither on a continuous scale nor when using the indicated cut-off value. Although this means that it is not an independent predictor of survival after surgery, it is currently the only parameter that can give an assessment of prognosis before surgery. We showed that dogs with a tumour diameter of ≥3 cm had significantly worse survival times after adrenalectomy than those with a diameter of <3 cm. Although previous studies reported that cut-off values of 2 cm10 or 5 cm22 should be used, classification based on these values did not result in significantly different survival times in our study. In some studies tumour size was not significantly associated with survival time.28,29 For future studies it might be use- ful to determine whether measurement of the tumour volume has additional prognostic value compared to measurement of the tumour diameter.
In this study we assessed whether the evaluated parameters can predict long-term survival. What we therefore did not assess was whether these parameters influence perioperative or short-term sur- vival. For example, although venous invasion as observed during imaging or surgery does not seem to affect long-term survival, as also reported in other studies,30,31 it could complicate the surgical procedure. Indeed, extensive invasion of the tumour into the caudal vena cava when the tumour thrombus extends beyond the hepatic hilus has been reported to increase perioperative mortality.31 In other studies, however, the presence of venous invasion did not affect perioperative mortality,10,30 so this possibly depends on the extent of invasion, the experience of the surgeon, and/or whether
cases with extensive venous invasion are considered to be candi- dates for surgery. Other factors that have been reported to increase perioperative mortality rates are acute adrenal haemorrhage and large tumours.30
When comparing the Utrecht score with scoring systems for human ACTs, it is noteworthy that it closely resembles the Helsinki score (3 x mitotic rate + 5 x necrosis + Ki67 PI).2 Because the intra- and inter-observer agreement scores for the assessment of mitotic figures were inadequate in our study, we did not include the mitotic rate in our survival analyses. The low number of HPFs that were analysed for the mitotic rate is a weakness of this study, because the agreement scores for the mitotic rate could possibly be improved by counting the number of mitotic figures in more HPFs. Interestingly, in both the Weiss and Weiss revisited score, clear cytoplasm in less than 25% of the tumour is a negative prognostic factor,3,6 similar to previ- ously described in canine ACTs,10 whereas in the Utrecht score more than 33% tumour cells with clear/vacuolated cytoplasm is a negative prognostic factor. Clear cytoplasm was, however, reported to be the least useful criterion in the Weiss score.6 In both the human and canine previous studies, cases with an ACT were selected irrespective of their hormonal status,3,6,10 whereas we included only cortisol- secreting ACTs. Whether this discrepancy in morphology of cytoplasm is related to a difference in assessment, interpretation or both, or to differences in the hormonal status of the ACT, remains to be elucidated.
A weakness of this study is its retrospective nature. Not all information had been documented consistently, for example, infor- mation on the tumour diameter was missing in four cases, and mea- surement of the tumour diameter was not standardized. Moreover, we were not able to reliably calculate tumour volumes because the multiple dimensions of the tumour were not often documented, nor was the tumour weight, which has prognostic value in human ACTs.9 The study’s retrospective nature could also have affected the reported survival times of the dogs, which were sometimes based on the owner’s estimate of when their dog had died. In addition, the reason for recurrence was not investigated in every case, so we could not make a distinction between local recurrence and recur- rence because of metastases.
Another remark is that although ACTs can be highly heteroge- neous, all histopathological parameters and the Ki67 PI have been assessed on just one tissue section of the ACT because of the retro- spective nature of this study. If the most malignant area of the tumour was not included in this tissue section, this could have resulted in an underestimation of the malignancy grade. However, even with just one tissue section the Utrecht score was able to distinguish three groups with significantly different survival times.
Although the fact that we only included parameters with intra- and inter-observer agreement scores of ≥0.4 improves the reliability of the scoring system, a drawback is that we may have excluded fac- tors that are important prognostic parameters. Moreover, the method used to create the Utrecht score has based its calculations on this specific subset of patients. To verify the validity of the score, a large prospective multi-institutional study in other patient groups and with the participation of more veterinary pathologists is required. Additionally, in this study we only included cortisol-secreting ACTs,
and whether the score can also be a valuable prognostic tool in, for example, non-secreting ACTs will have to be determined in future studies.
In conclusion, we introduce the Utrecht score to post-surgically assess the prognosis of dogs with cortisol-secreting ACTs. Having an accurate assessment of prognosis is useful to communicate to the dog’s owner, but could also assist in selecting high-risk dogs that might benefit from additional monitoring, adjuvant therapy with, for example, mitotane, or both. Moreover, a solid histopathological scor- ing system represents a basis for future studies on other markers of malignancy, which could facilitate the identification of potential future treatment targets.
ACKNOWLEDGEMENTS
The authors would like to thank Jolle Kirpensteijn, Elaine Naan and Bart Sjollema for their surgical contributions to our dataset; Ellen Deelen and Adri Slob for optimization of the Ki67 staining protocol; and Tim van Olmen and the Veterinary Pathology Diagnostic Centre for technical assistance.
CONFLICT OF INTEREST
The authors have no conflict of interest to report.
ORCID
Karin Sanders [ https://orcid.org/0000-0001-9634-9853 Jan A. Mol (D https://orcid.org/0000-0002-0843-8088 Hans S. Kooistra ID https://orcid.org/0000-0002-8058-0492
Sara Galac ID https://orcid.org/0000-0002-4831-4995
REFERENCES
1. Galac S, Reusch CE, Kooistra HS, Rijnberk A. Adrenals. In: Rijnberk A, Kooistra HS, eds. Clinical Endocrinology of Dogs and Cats. 2nd ed. Hannover, Germany: Schlütersche; 2010:93-154.
2. Pennanen M, Heiskanen I, Sane T, et al. Helsinki score - a novel model for prediction of metastases in adrenocortical carcinomas. Hum Pathol. 2015;46(3):404-410.
3. Aubert S, Wacrenier A, Leroy X, et al. Weiss system revisited: a clini- copathologic and immunohistochemical study of 49 adrenocortical tumors. Am J Surg Pathol. 2002;26(12):1612-1619.
4. Blanes A, Diaz-Cano SJ. Histologie criteria for adrenocortical prolifera- tive lesions: value of mitotic figure variability. Am J Clin Pathol. 2007; 127(3):398-408.
5. van Slooten H, Schaberg A, Smeenk D, Moolenaar AJ. Morphologic characteristics of benign and malignant adrenocortical tumors. Cancer. 1985;55(4):766-773.
6. Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol. 1984;8(3): 163-169.
7. Weiss LM, Medeiros LJ, Vickery AL. Pathologic features of prognostic significance in adrenocortical carcinoma. Am J Surg Pathol. 1989;13(3): 202-206.
8. Duregon E, Cappellesso R, Maffeis V, et al. Validation of the prognos- tic role of the “Helsinki score” in 225 cases of adrenocortical carci- noma. Hum Pathol. 2017;62:1-7.
9. Papotti M, Libè R, Duregon E, Volante M, Bertherat J, Tissier F. The Weiss score and beyond-histopathology for adrenocortical carcinoma. Horm Cancer. 2011;2(6):333-340.
10. Labelle P, Kyles AE, Farver TB, de Cock HEV. Indicators of malignancy of canine adrenocortical tumors: histopathology and proliferation index. Vet Pathol. 2004;41(5):490-497.
11. Morimoto R, Satoh F, Murakami O, et al. Immunohistochemistry of a proliferation marker Ki67/MIB1 in adrenocortical carcinomas: Ki67/MIB1 labeling index is a predictor for recurrence of adrenocorti- cal carcinomas. Endocr J. 2008;55(1):49-55.
12. Schmitt A, Saremaslani P, Schmid S, et al. IGFII and MIB1 immuno- histochemistry is helpful for the differentiation of benign from malignant adrenocortical tumours. Histopathology. 2006;49(3): 298-307.
13. Soon PSH, Gill AJ, Benn DE, et al. Microarray gene expression and immunohistochemistry analyses of adrenocortical tumors identify IGF2 and Ki-67 as useful in differentiating carcinomas from adenomas. Endocr Relat Cancer. 2009;16(2):573-583.
14. Wachenfeld C, Beuschlein F, Zwermann O, et al. Discerning malig- nancy in adrenocortical tumors: are molecular markers useful? Eur J Endocrinol. 2001;145(3):335-341.
15. Fassnacht M, Johanssen S, Quinkler M, et al. Limited prognostic value of the 2004 international union against cancer staging classi- fication for adrenocortical carcinoma. Cancer. 2009;115(2): 243-250.
16. Beuschlein F, Weigel J, Saeger W, et al. Major prognostic role of Ki67 in localized adrenocortical carcinoma after complete resection. J Clin Endocrinol Metab. 2015;100(3):841-849.
17. Jouinot A, Bertherat J. Adrenocortical carcinoma: differentiating the good from the poor prognosis tumors. Eur J Endocrinol. 2018;178(5): R215-R230.
18. Behrend EN, Kooistra HS, Nelson R, Reusch CE, Scott-Moncrieff JC. Diagnosis of spontaneous canine hyperadrenocorticism: 2012 ACVIM consensus statement (small animal). J Vet Intern Med. 2013;27(6): 1292-1304.
19. Fuhrman SA, Lasky LC, Limas C. Prognostic significance of morpho- logic parameters in renal cell carcinoma. Am J Surg Pathol. 1982;6(7): 655-663.
20. Schneider CA, Rasband WS, Eliceiri KW. NIH image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671-675.
21. Landis JR, Koch GG. The measurement of observer agreement for cat- egorical data. Biometrics. 1977;33(1):159-174.
22. Massari F, Nicoli S, Romanelli G, Buracco P, Zini E. Adrenalectomy in dogs with adrenal gland tumors: 52 cases (2002-2008). J Am Vet Med Assoc. 2011;239(2):216-221.
23. Papathomas TG, Pucci E, Giordano TJ, et al. An international Ki67 reproducibility study in adrenal cortical carcinoma. Am J Surg Pathol. 2016;40(4):569-576.
24. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646-674.
25. Ovalle WK, Nahirney PD. The cell. Netter’s Essential Histology. Phila- delphia, PA: Elsevier; 2008:1-29.
26. Margonis GA, Kim Y, Tran TB, et al. Outcomes after resection of cortisol- secreting adrenocortical carcinoma. Am J Surg. 2016;211(6):1106-1113.
27. Else T, Williams AR, Sabolch A, Jolly S, Miller BS, Hammer GD. Adju- vant therapies and patient and tumor characteristics associated with survival of adult patients with adrenocortical carcinoma. J Clin Endocrinol Metab. 2014;99(2):455-461.
28. Anderson CR, Birchard SJ, Powers BE, Belandria GA, Kuntz CA, Withrow SJ. Surgical treatment of adrenocortical tumors: 21 cases (1990-1996). J Am Anim Hosp Assoc. 2001;37:93-97.
29. Schwartz P, Kovak JR, Koprowski A, Ludwig LL, Monette S, Bergman PJ. Evaluation of prognostic factors in the surgical treatment of adrenal gland tumors in dogs: 41 cases (1999-2005). J Am Vet Med Assoc. 2008;232(1):77-84.
30. Lang JM, Schertel E, Kennedy S, Wilson D, Barnhart M, Danielson B. Elective and emergency surgical management of adrenal gland tumors: 60 cases (1999-2006). J Am Anim Hosp Assoc. 2011;47(6):428-435.
31. Barrera JS, Bernard F, Ehrhart EJ, Withrow SJ, Monnet E. Evaluation of risk factors for outcome associated with adrenal gland tumors with or without invasion of the caudal vena cava and treated via adrenalectomy in dogs: 86 cases (1993-2009). J Am Vet Med Assoc. 2013;242(12):1715-1721.
SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of this article.
How to cite this article: Sanders K, Cirkel K, Grinwis GCM, et al. The Utrecht Score: A novel histopathological scoring sys- tem to assess the prognosis of dogs with cortisol-secreting adrenocortical tumours. Vet Comp Oncol. 2019;17:329-337. https://doi.org/10.1111/vco.12474