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Prognostic Value of Microscopic Tumor Necrosis in Adrenal Cortical Carcinoma
Michaela Luconi1,2,3 . Giulia Cantini1,2,3 . Rachel S. van Leeuwaarde4 . Rogina Roebaar4 . Laura Fei1 . Arianna Pia Propato1 . Raffaella Santi5 . Tonino Ercolino6 . Massimo Mannelli1,2,3 . Letizia Canu1,2,3 . Ronald R. de Krijger7,8 . Gabriella Nesi2,3,5
Accepted: 8 March 2023 / Published online: 23 March 2023 @ The Author(s) 2023
Abstract
Adrenal cortical carcinoma (ACC) is an uncommon neoplasm with variable prognosis. Several histologic criteria have been identified as predictors of malignancy in adrenal cortical tumors. The Weiss score is the system most widely employed for diagnostic purposes, but also possesses prognostic value. We aim to determine the relative impact of each Weiss parameter on ACC patient survival. A multicenter retrospective analysis was conducted on a series of 79 conventional ACCs surgically treated at the Florence and Utrecht centers of the European Network for the Study of Adrenal Tumors (ENSAT). Weiss clas- sification was recapitulated using principal component analysis (PCA). The Kaplan-Meier and Cox multivariate regression analyses were applied in order to estimate the prognostic power of Weiss versus other clinical parameters. PCA reduced the nine Weiss parameters to the best fitting 4-component model, each parameter clustering with a single component. Necrosis and venous invasion clustered together with the highest scores, thus establishing the most relevant component (Component 1) to explain Weiss distribution variability. Only Component 1 significantly predicted overall survival (OS, log-rank =0.008) and disease-free survival (DFS, log-rank <0.001). When considering the prognostic power of Weiss parameters, necrosis alone could independently assess OS (log-rank =0.004) and DFS (log-rank <0.001) at both the Kaplan-Meier and multi- variate Cox regression analyses [hazard ratio (HR)=7.8, 95% confidence interval [CI]= 1.0-63.5, p=0.05, and HR = 12.2, 95% CI=1.6-95.0, p=0.017, respectively]. The presence of necrosis significantly shortened time to survival (TtS) and time to recurrence (TtR), 57.5 [31.5-103.5] vs 34 [12-78] months (p=0.05) and 57.5 [31.5-103.5] vs 7 [1.0-31.5] months (p<0.001), respectively. Our study suggests that, of the Weiss parameters, necrosis is the most powerful adverse factor and the best predictor of OS and DFS in ACC patients.
Keywords Weiss score . ACC . Survival analysis . Principal component analysis
Introduction
Adrenal cortical carcinoma (ACC) is a rare and often aggres- sive malignancy, with a 5-year survival less than 20% in patients with metastatic disease [1]. In addition to conventional
clinical factors such as tumor size, stage, functional activity, total Weiss score, and Ki67 proliferation index, a number of molecular markers have recently been recognized to improve accuracy for survival prediction [2-5], particularly in meta- static cases [6, 7]. However, implementation of validated
☒ Michaela Luconi michaela.luconi@unifi.it
☒ Gabriella Nesi gabriella.nesi@unifi.i
1 Endocrinology Unit, Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale Gaetano Pieraccini 6, Florence 50139, Italy
2 Centro di Ricerca E Innovazione Sulle Patologie Surrenaliche, AOU Careggi, 50134 Florence, Italy
3 ENS@T Center of Excellence, Florence, Italy
4 Department of Endocrine Oncology, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands
5 Pathology Section, Department of Health Sciences, University of Florence, Viale Gaetano Pieraccini 6, Florence 50139, Italy
6 Endocrinology Unit, Careggi University Hospital (AOUC), Florence 50139, Italy
7 Princess Maxima Center for Pediatric Oncology, Utrecht 3584 CS, The Netherlands
8 Department of Pathology, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands
histologic parameters may also be of use in identifying patients for correct stratification and individualized follow-up.
The Weiss score is the most widely applied histologic multiparameter scoring system for the classification of adrenal cortical tumors in adults. It is based on 9 histo- logic criteria, with the inherent problems of subjective scoring and intra- and interobserver variability. While endorsed in the most recent WHO blue book on endocrine and neuroendocrine tumors, the fact that other, newer, systems have been developed, such as the Helsinki score and reticulin algorithm, indicates room for improvement [8-10]. Importantly, not all 9 parameters may carry the same diagnostic and prognostic relevance, but so far no studies have addressed this issue [5].
Principal component analysis (PCA) is a statistical tool applicable to large datasets for reducing the number of correlated variables, increasing interpretability, but at the same time minimizing information loss [11]. This is achieved by a linear transformation of the initial dataset into new uncorrelated variables that in turn maximize vari- ance. The new variables, named “principal components,” are not generated a priori, rendering PCA an adaptive data analysis technique.
The current study aimed to evaluate the prognostic value of individual Weiss parameters in a large ACC data- set, in an attempt to simplify and prioritize their analysis adopting a PCA approach.
Materials and Methods
Patients and Ethical Approval
We retrospectively analyzed a series of 79 conventional ACCs following surgical removal of the tumor at Careggi University Hospital, Florence, Italy (n=57) or Univer- sity Medical Center Utrecht, Utrecht, The Netherlands (n=22) between 1999 and 2021. Formalin-fixed paraffin- embedded tumor samples were available for immunohis- tochemical analysis. The study design was reviewed and approved by the Careggi University Hospital Ethical Com- mittee (Prot. 2011/0020149 - Rif CEAVC Em. 2019-201 26/11/2019). The Medical Ethical Committee (MERC) of the University Medical Center Utrecht confirmed that the Medical Research Involving Human Subjects Act (WMO) did not apply to this study and no official approval was required under WMO. The patients recruited in Florence gave their written informed consent, which was not neces- sary for the Dutch cohort.
Pathologic Analysis of ACC Samples
Histologic diagnosis of ACC was carried out by two inde- pendent reference pathologists on tumor tissue removed
at surgery. Tumor specimens were evaluated according to the Weiss scoring system, in which the presence of three or more criteria highly correlates with malignant behavior [12]. The Ki67 labeling index (LI) was estimated on digitalized glass slides, after immunohistochemical staining with anti- human Ki67 antibody (1:40 dilution, MIB-1, Dako, Carpin- teria, CA, USA). Areas with the highest labeling were manu- ally identified, Ki67-positive nuclei were counted in 1000 tumor cells, and Ki67 LI was expressed as the percentage of labeled cells, using the Ki67 algorithm available in the Pic- ture Archiving and Communication System (PACS) (Sectra Medical, Linkoping, Sweden). Tumor stage was assessed according to the revised 8th edition of the TNM classifica- tion of ACC proposed by ENSAT [13].
Statistical Analysis
Continuous variables with normal distribution were pre- sented as mean (standard deviation [SD]) and non-normal variables as median (interquartile range [IQR]). Categorical variables were expressed as counts and percentages. Sta- tistical analysis was performed with SPSS 26.0 (Statistical Package for the Social Sciences, Chicago, IL, USA) for Windows. A p-value less than 0.05 was considered statis- tically significant. Possible associations were investigated using the x2 test for categorical variables and Pearson’s cor- relation for continuous variables. Comparison between two groups of data was accomplished using Student’s t-test for normally distributed variables and U Mann-Whitney test for non-parametric variables. Overall survival (OS) and disease- free survival (DFS) are defined as the probability (ranging from 0 to 1) that a patient diagnosed with the disease is still alive (OS) or free from the disease (DFS) at a time point from surgery. Survival analysis was estimated through the Kaplan-Meier method, and differences between groups were calculated by the log-rank test. Only variables significantly predicting OS and DFS at the Kaplan-Meier analysis were used for univariate and multivariate Cox regression analyses to define hazard ratio (HR) and 95% confidence intervals (CI). PCA was performed on the ACC cases to ascertain the best model for clustering and reducing the number of Weiss parameters (i.e., diffuse architecture, confluent necrosis, clear cells comprising less than 25% of the tumor, venous invasion, sinusoidal invasion, capsular invasion, high nuclear grade, mitotic count of > 5 mitoses per 50 high-power fields (10 mm2), and atypical mitotic figures) in an unsupervised and un-hierarchical manner.
Results
The main clinico-pathologic characteristics of ACC patients (n=79) are illustrated in Table 1. Our cohort comprised 52 (64%) female and 27 (34%) male patients, with a mean + SD
age of 49.2 + 15.1 years. Increased hormonal secretion was observed in the majority of patients (n=47/75; 59%) with glucocorticoid excess being the most frequent aberra- tion (n=32/47; 68%). There were 28 (35%) non-secreting tumors, and for 4 patients relevant data were not available. Tumor stage was I in 12 (15%) patients, II in 30 (38%), III in 20 (25%), and IV in 17 (22%). Mean ± SD tumor size was 11.2±5.9 cm. Among the 69 patients for whom resection margin status (RMS) was known, 44 (56%) had undergone complete surgical resection (R0). Median [IQR] Weiss score was 6 [5-7]; distribution of each Weiss parameter in our cohort is detailed in Table 2. In particular, necrosis was a frequent finding, observed in 61 (77%) patients (Fig. 1). Mean + SD Ki67 LI was 27.3+25.7. Median [IQR] follow- up was 39 [16-82] months and time to recurrence (TtR) 11 [3-59] months. Overall, 29 (37%) patients died of the disease and 42 (53%) experienced recurrence.
| Variable | Values |
|---|---|
| Age (years) | 49.2±15.1 (range 10-79) |
| Sex | F: 52 (64%) |
| M: 27 (34%) | |
| Secretion | NS: 28 (35%) |
| GC: 32 (41%) | |
| A: 14 (18%) | |
| MC: 1 (1%) | |
| NA: 4 (5%) | |
| Tumor size (cm) | 11.2±5.9 (range 2-30) |
| Stage | I: 12 (15%) |
| II: 30 (38%) | |
| III: 20 (25%) | |
| IV: 17 (22%) | |
| Total Weiss score | 6.0 [5-7] (range 3-9) |
| Ki67 LI | 27.3±25.7 (range 2-96) |
| RMS | R0: 44 (56%) |
| R1: 25 (32%) | |
| NA: 10 (12%) | |
| Recurrence | Y: 42 (47%) |
| N: 37 (53%) | |
| Death | Y: 29 (37%) |
| N: 50 (63%) | |
| FU (months) | 39 [16-82] (range 1-258) |
| TtR (months) | 11 [3-59-82] (range 0-258) |
FU follow-up time, TtR time to recurrence, NS non-secreting, GC glucocorticoids, A androgens, MC mineralocorticoids, NA not avail- able, LI labeling index, RMS resection margin status, Y yes, N no
PCA of the nine Weiss parameters, conducted stepwise from an autovalue > 1, identified a 4-component model with a cumulative variance of 60%. Direct Varimax rotation was then applied to ensure the best fitting clustering and optimi- zation of parameter variance, mostly attributable to a single component for each parameter (Table 3). Component 1, com- bining necrosis and venous invasion, accounted for almost 25% of the Weiss score variance. To assess the predictive value of the four components, we evaluated OS and DFS by the Kaplan-Meier analysis. When stratifying patients into three classes according to the Weiss parameters clustered in each component, only Component 1 significantly predicted OS (log-rank=0.008) and DFS (log-rank <0.001) (Fig. 2).
To determine which parameters best correlated with death and recurrence, we performed regression analysis on continuous variables, i.e., age, tumor stage, tumor size, Weiss score, and Ki67 LI, as well as dichotomized vari- ables, such as presence or absence of necrosis and venous invasion, grouped tumor stage (I-II vs III-IV), tumor size (cut-off=6 cm), RMS (R0 vs R1), total Weiss score (cut- off value =6), and Ki67 LI (cut-off value =20%) (Table 4). Of the Weiss parameters, necrosis alone was significantly associated with death (r/x2=9.7, p<0.002) and recurrence (r/x2=21.2, p<0.001), while venous invasion correlated with recurrence only (rlx2=10.7, p=0.001). In order to test whether RMS could override microscopic features in rela- tion to outcome, we re-ran regression analysis on completely resected tumors (R0). Necrosis remained the only histologic factor related to both relapse and death (Table 4).
We therefore focused on necrosis to estimate its pre- dictive power for survival. Necrosis was identified in 61
| Weiss parameter | Total cohort (n=79) | R0 patients (n=44) | ||
|---|---|---|---|---|
| Negative n (%) | Positive n (%) | Negative n (%) | Positive n (%) | |
| High nuclear grade | 19 (24%) | 60 (76%) | 13 (30%) | 31 (70%) |
| Atypical mitosis | 42 (53%) | 37 (47%) | 28 (64%) | 16(36%) |
| > 5 mitoses per 50 | 13 (16%) | 66 (84%) | 9 (20%) | 35 (80%) |
| high-power fields (10 mm2) | ||||
| Clear cells ≤25% | 11 (14%) | 68 (86%) | 4 (9%) | 40 (91%) |
| Diffuse architecture | 22 (28%) | 57 (72%) | 8 (18%) | 36 (82%) |
| Necrosis | 18 (23%) | 61 (77%) | 13 (30%) | 31 (70%) |
| Venous invasion | 40 (51%) | 39 (49%) | 24 (54%) | 20 (46%) |
| Sinusoidal invasion | 38 (48%) | 41 (52%) | 21 (48%) | 23 (52%) |
| Capsular invasion | 35 (44%) | 44 (56%) | 18 (41%) | 26 (59%) |
a
b
(77%) of all cases, occurring in 47/61 (77%) of tumors with Weiss score ≥6 and in 14/61 (23%) with Weiss score <6 (rlx2=21.8, p<0.001), with a higher prevalence in masses measuring ≥6 cm than in those < 6 cm (92% vs 8%, respec- tively; r/x2=24.1,p<0.001). Necrosis was present in 27/61 (44%) low stage (I-II) cases and absent in 3/18 (17%) high stage (III-IV) cases (r/x2=6.2, p=0.012). Tumors with
necrosis also displayed higher Ki67 LI and a higher risk of death and recurrence than tumors without necrosis; only one death (1/18; 6%) and one recurrence (1/18; 6%) were recorded in cases without microscopic necrosis. In the R0 subgroup, the percentage of tumors without necrosis was higher than in the whole cohort (29.5% and 22.8%, respec- tively), while necrosis retained its significance with recur- rence, death, and TtR (Table 5).
The impact of each Weiss variable on OS and DFS was assessed by the Kaplan-Meier analysis. Necrosis alone proved to significantly predict OS (log-rank=0.004, Fig. 3A) and DFS (log-rank <0.001, Fig. 3B), with results compara- ble to those of dichotomized tumor stage (Fig. 3C and D) and size (Fig. 3E and F). Venous invasion significantly pre- dicted DFS (log-rank=0.002), but not OS (log-rank=0.057, Fig. 3G and H). By stratifying ACC patients for dichotomized Ki67 LI (cut-off value =20%), the Kaplan-Meier curves revealed weak prognostic power (OS, log-rank =0.031; DFS, log-rank =0.088). Significant differences in time to survival (TtS) and TtR were recorded only when grouping patients for the presence or absence of necrosis (34 [12-78] vs 57.5 [31.5-103.5] months, p=0.05 for TtS and 7 [1.0-31.5] vs 57.5 [31.5-103.5] months, p<0.001 for TtR). No signifi- cant differences in the OS and DFS curves were observed for the other Weiss parameters (data not shown). When the Kaplan-Meier analysis was performed in RO patients, necro- sis remained significant for both OS and DFS (Fig. 3I and J), while all other variables (stage, size, venous invasion) lost their predictive value (data not shown).
Finally, we estimated the risk of recurrence and death by multivariate Cox regression analysis (Table 6). Necro- sis, considered separately or combined with venous invasion as Component 1, retained significance in predicting risk of death and recurrence after adjustment for age, tumor size and total Weiss score.
| Weiss parameter | Component 1 | Component 2 | Component 3 | Component 4 |
|---|---|---|---|---|
| Necrosis | 0.801 | |||
| Venous invasion | 0.744 | |||
| Sinusoidal invasion | 0.808 | |||
| Capsular invasion | 0.654 | |||
| Diffuse architecture | 0.646 | |||
| Atypical mitosis | 0.879 | |||
| > 5 mitoses per 50 high-power fields (10 mm2) | 0.657 | |||
| Nuclear atypia | 0.590 | |||
| Clear cells ≤ 25% | 0.836 |
Data are expressed as correlation values between parameters and components> 0.450, and are indicated in descending order. Barlett’s test value for adequacy =0.548 and significance of the correlation matrix p<0.001. Descending weight of the single components from Component 1 to 4 for covering variance of Weiss score
OS
A
1,0
n=16
555
0,8
necrosis or venus invasion +
Overall Survival
0,6
0,4
n=37
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n=26
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Log rank=0.008
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Overall Survival Time (months)
necrosis & venus invasion, both -
necrosis & venus invasion, both +
Discussion
The high mortality and limited therapeutic options cur- rently available for ACC require accurate patient stratifi- cation for deciding on personalized and precise treatment [14-16]. In a two ENSAT center-based ACC cohort of 79 patients, we applied PCA to Weiss parameters to reduce the number of non-correlated variables and evaluate their relative weight. We also analyzed the prognostic value of individual Weiss score criteria in combination with more traditional variables (e.g., tumor size, TNM stage, margin status). Only necrosis and its relevant PCA component proved to significantly predict patient survival, also retain- ing its prognostic ability in the R0 subgroup.
To the best of our knowledge, this is the first applica- tion of an un-hierarchical PCA approach to simplify and prioritize Weiss score parameters. Coste et al. previously used Rasch model analysis to assess the unidimensional- ity of the Weiss score, concluding that the biases gener- ated by differential item functioning (DIF) due to the size and weight of the tumor for two items (diffuse architec- ture and necrosis) may be substantial [17]. However, the complementary factor analyses they conducted was not un-hierarchical and was run on three pre-fixed domains (nucleus, architecture, and invasion), thus forcing specific association between parameters [17].
In our study, PCA was unrestrained and grouped the nine parameters into four rather than three independent compo- nents, with each parameter unequivocally related to one specific component. Differently from Coste et al., necrosis
DFS
B
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n=16
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Disease Free Time (months)
1. Statistically significant differences in the Kaplan-Meier analysis were evaluated as log-rank; number of patients is indicated
did not cluster in the architecture domain, but combined with venous invasion in Component 1. We can speculate that this finding may be relevant to tumor metastatic poten- tial. Intratumoral hypoxia, histologically reflected by the presence of necrosis, can lead to the development of an aggressive phenotype, which includes cell immortalization and dedifferentiation, pH regulation, autocrine growth/sur- vival, angiogenesis, and invasion/metastasis.
Lymphatic (sinusoidal) and capsular invasion clustered with diffuse tumor architecture in Component 2, while Component 3 included the cytologic items (high nuclear grade, high mitotic rate, and atypical mitoses). Finally, Component 4 comprised the low prevalence of lipid- rich or clear cells in the tumor mass (≤25%), a marker of adrenal cortical differentiation. Cell lipid content and quality have recently been suggested to be highly relevant for shaping cancer cell phenotype [18], and Sterol O-Acyl Transferase 1 (SOAT1), a key enzyme for cholesterol esterification, has been established as an independent predictor of death and recurrence in ACC patients [19]. The reciprocal crosstalk lately demonstrated between adipose-derived stem cells and the ACC cell line H295R confirms the relevance of lipid metabolism in cancer cell reprogramming and invasion [20, 21]
Interestingly, Component 1 was not only the main meas- ure of Weiss distribution variability at PCA, but also the only component capable of significantly predicting OS and DFS at the Kaplan-Meier analysis. In our series, necrosis was not detected in all tumors, being present in 70% of low stages (I-II) and absent in 11% of high stages (III-IV). This
| Variable | Total cohort (n =79) | R0 patients (n=44) | ||
|---|---|---|---|---|
| Death rl2 | Recurrence r12 | Death rlx | Recurrence rlx | |
| Necrosis | 9.7 | 21.2 | 4.7 | 10.5 |
| 0.002 | <0.001 | 0.029 | 0.001 | |
| Venous invasion | 3.0 | 10.7 | 2.1 | 5.5 |
| 0.085 | 0.001 | 0.152 | 0.019 | |
| RMS | 11.0 | 14.5 | - | - |
| (R0/R1) | <0.001 | <0.001 | ||
| Age | 0.256 | 0.239 | 0.261 | 0.411 |
| 0.024 | 0.034 | 0.086 | 0.006 | |
| Tumor size | 0.102 | 0.317 | -0.063 | 0.192 |
| 0.376 | 0.004 | 0.687 | 0.211 | |
| Tumor stage | 0.343 | 0.580 | 0.080 | 0.448 |
| 0.002 | <0.001 | 0.605 | 0.002 | |
| Total Weiss score | 0.106 | 0.273 | 0.064 | 0.211 |
| 0.352 | 0.015 | 0.682 | 0.170 | |
| Ki67 LI | 0.157 | 0.186 | 0.195 | 0.008 |
| 0.256 | 0.179 | 0.205 | 0.960 | |
| Dichotomized tumor size | 5.1 | 17.7 | 1.5 | 9.4 |
| (cut-off 6 cm) | 0.024 | <0.001 | 0.222 | 0.002 |
| Grouped tumor stage | 5.6 | 15.8 | 0.45 | 4.3 |
| (I-II vs III-IV) | 0.018 | 0.000 | 0.832 | 0.038 |
| Dichotomized Weiss score | 1.6 | 2.6 | 0.134 | 0.6 |
| (cut-off 6) | 0.200 | 0.110 | 0.714 | 0.447 |
| Dichotomized Ki67 LI | 1.3 | 1.9 | 0.049 | 0.052 |
| (cut-off 20%) | 0.248 | 0.172 | 0.825 | 0.820 |
Necrosis, venous invasion, and resection margin status (RMS) are dichotomized variables (presence or absence); age, tumor size, tumor stage, total Weiss score, and Ki67 labeling index (LI) are considered con- tinuous variables; grouped tumor stage and dichotomized Weiss score, tumor size, and Ki67 LI are non-con- tinuous variables; r/x2 is reported for continuous and categorical variables, respectively; p values are indicated (significant in italics)
finding further underlines the prognostic value of histologic coagulative tumor necrosis, comparable to conventional parameters such as tumor stage (I-II vs III-IV) and size (cut-off 6 cm).
Ki67 proliferation index has been suggested to be a strong prognostic indicator in both localized and advanced ACC [1, 22]. However, the Ki67 scoring assessment may differ con- siderably, and both inter- and intra-observer variability poses limitations to its clinical utility. In our study, Ki67 index revealed poor predictive power (DFS: log-rank=0.088; OS: log-rank =0.031 for cut-off 20%), probably due to the high proportion of missing values. Despite our efforts to revise the Ki67 LI for all patients, we were able to retrieve only 26 (33%) of 79 tumor samples for immunohistochemical examination and centralized revision.
We acknowledge some limitations of our study: (i) the ret- rospective nature of the study and long recruitment period, which might have affected variance in some analyses; (ii) incomplete dataset for Ki67 scoring assessment, with only one-third of values available; (iii) the absence of a validation cohort. The main strengths of the study are (i) the relatively large cohort of patients from two independent ENSAT centers, (ii) the good quality of our data, and (iii) the un-hierarchical PCA approach employed.
In conclusion, PCA applied to the Weiss scoring sys- tem suggests that necrosis is the most powerful adverse factor and the best histologic predictor of OS and DFS in ACC. Future prospective trials with centralized patho- logic evaluation should be conducted to validate these preliminary results.
| Variable | Total cohort (n=79) | R0 patients (n=44) | ||||
|---|---|---|---|---|---|---|
| Absence necrosis (n=18) | Presence necrosis (n =61) | P (X) | Absence necrosis (n=13) | Presence necrosis (n=31) | P (X) | |
| Age | 46.8±16.9 | 46.9±14.6 | 0.444 | 47.9±15.4 | 49.6±13.2 | 0.720 |
| Sex (F:M) | 11:7 | 41:20 | 0.631 (0.230) | 7:6 | 19:12 | 0.647 (0.210) |
| Secretion | 0.139 (6.9) | 0.441 (3.7) | ||||
| N | 7 | 21 | 5 | 6 | ||
| GC | 4 | 28 | 3 | 10 | ||
| A | 5 | 9 | 3 | 6 | ||
| MC | 1 | 0 | 1 | 0 | ||
| NA | 1 | 3 | 0 | 0 | ||
| Tumor size (cm) | 7.3±6.5 | 12.4±5.2 | <0.001 | 7.2±7.5 | 12.0±5.2 | 0.020 |
| Stage | <0.001 (23.3) | <0.001 (19.3) | ||||
| I | 9 | 3 | 8 | 1 | ||
| II | 6 | 24 | 3 | 16 | ||
| III | 2 | 18 | 2 | 12 | ||
| IV | 1 | 16 | 0 | 2 | ||
| Total Weiss score | 4.2±1.3 | 6.7±1.4 | <0.001 | 4.3±1.4 | 6.4±1.3 | <0.001 |
| Ki67 LI | 8.6±6.6 | 31.6±26.5 | <0.001 | 11.1±11.2 | 27.4±23.5 | 0.003 |
| RMS | 0.037 (4.3) | - | - | - | ||
| R0 | 13 | 31 | ||||
| R1 | 2 | 23 | ||||
| NA | 3 | 8 | ||||
| Recurrence | <0.001 (21.2) | 0.001 (10.6) | ||||
| N | 17 | 20 | 13 | 15 | ||
| Y | 1 | 41 | 0 | 16 | ||
| Death | 0.002 (9.7) | 0.029 (4.7) | ||||
| N | 17 | 33 | 13 | 22 | ||
| Y | 1 | 28 | 0 | 9 | ||
| TtS (months) | 57.5 [31.5-103.5] | 34 [12-78] | 0.050 | 56 [37-95] | 42 [22-88] | 0.418 |
| TtR (months) | 57.5 [31.5-103.5] | 7 [1.0-31.5] | <0.001 | 56 [37-95] | 25 [4-70] | 0.013 |
| Dichotomized tumor size (cut-off 6 cm) | <0.001 (24.1) | <0.001 (16.4) | ||||
| < | 11 | 5 | 9 | 3 | ||
| ≥ | 7 | 56 | 4 | 28 | ||
| Tumor stage | 0.012 (6.2) | 0.060 (3.5) | ||||
| I-II | 15 | 27 | 11 | 17 | ||
| III-IV | 3 | 34 | 2 | 14 | ||
| Dichotomized Weiss score (cut-off 6) | <0.001 (21.8) | <0.001 (11.4) | ||||
| < | 15 | 14 | 10 | 7 | ||
| ≥ | 3 | 47 | 3 | 24 | ||
Data are expressed as mean+SD and frequency for continuous and categorical variables, respectively; TtS and TtR are expressed as median [interquartile]; p values are indicated (significant in italics) for Student’s t-test analysis for continuous parametric variables, U Mann-Whitney test for continuous non-parametric or for x2 test (in brackets) for categorical variables
NS non-secreting, GC glucocorticoids, A androgens, MC mineralocorticoids, NA not available, LI labeling index, RMS resection margin status, N no, Y yes, TtS time to survival (TtS), TtR time to recurrence
A
OS
necrosis
B
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necrosis
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Overall Survival
n=18
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n=18
0,8
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0,6
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C
1,0
stage III-IV
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n=41
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n=38
n=38
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E
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1,0
diameter ≥ 6
1,0
diameter < 6
n=16
0,8
0,8
Overall Survival
n=16
Disease Free Survial
0,6
0,6
0,4
0,4
n=63
0,2
-n=63
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0,0
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225
250
275
300
1,0
1,0
venus invasion +
0,8
venus invasion
0,8
Overall Survival
Disease Free Survial
0,6
0,6
n=40
0,4
0,4
n=39
n=40
0,2
0,2
0,0
Log rank=0.057
0,0
n=39
Log rank=0.002
0
25
50
75
100
125
150
175
200
225
250
275
300
0
25
50
75
100
125
150
175
200
225
250
275
300
I
J
1,0
1,0
n=13
n=13
necrosis
+
0,8
necrosis
Disease Free Survial
0,8
Overall Survival
0,6
0,6
n=31
0,4
0,4
0,2
0,2
0,0
Log rank=0.050
n=31
0,0
Log rank=0.002
0
20
40
60
80
100
120
140
160
180
0
20
40
60
80
100
120
140
Overall Survival Time (months)
Disease Free Survival Time (months)
4Fig. 3 Comparison between survival predictive value of different parameters in the ACC cohort. The Kaplan-Meier analysis of OS and DFS in ACC patients stratified into two classes according to the presence of necrosis (A, B), tumor stage (C, D), venous invasion (E, F) tumor size (G, H, diameter <or≥6 cm). Necrosis maintained its predictive power for both OS (I) and DFS (J) in the R0 subgroup. Statistically significant differences in the Kaplan-Meier analysis were evaluated as log-rank; number of patients is indicated
| Variable | Recurrence Death | |||||
|---|---|---|---|---|---|---|
| HR | 95% CI | p | HR | 95% CI | p | |
| Necrosis | 12.2 | [1.6-95.0] | 0.017 | 7.8 | [1.0-63.5] | 0.050 |
| Venous invasion | 0.6 | [0.6-2.16] | 0.588 | 1.0 | [0.4-2.5] | 0.974 |
| Age | 1.0 | [1.0-1.1] | 0.011 | 1.0 | [1.0-1.1] | 0.014 |
| Tumor size | 1.0 | [1.0-1.1] | 0.149 | 1.0 | [0.9-1.1] | 0.615 |
| Total Weiss score | 1.1 | [0.8-1.4] | 0.549 | 1.1 | [0.8-1.5] | 0.551 |
| Recurrence | Death | |||||
|---|---|---|---|---|---|---|
| HR | 95% CI | p | HR | 95% CI | p | |
| Component 1 | 1.8 | [1.1-3.0] | 0.016 | 2.1 | [1.1-4.1] | 0.022 |
| Age | 1.1 | [1.0-1.1] | 0.004 | 1.0 | [1.0-1.1] | 0.003 |
| Tumor size | 1.1 | [1.0-1.1] | 0.010 | 1.0 | [1.0-1.1] | 0.507 |
| Total Weiss score | 1.3 | [1.0-1.7] | 0.014 | 1.3 | [1.0-1.7] | 0.050 |
Hazard ratio (HR) and 95% confidence intervals (CI) are indicated for multivariate analysis; p values are indicated (significant in italics)
Author Contribution M.L. and G.N. wrote the main manuscript text; G.C., R.R., L.F., A.P.P., R.S., T.E., and L.C. collected and analyzed data; R.R.K, R.S.L., M.M., M.L., and G.N. supervised the project. All authors approved the final version of the manuscript.
Funding Open access funding provided by Università degli Studi di Firenze within the CRUI-CARE Agreement. This study was supported by Associazione Italiana Ricerca sul Cancro (AIRC) Investigator Grant to M. L. (grant # IG2015-17691).
Availability of Data and Materials The datasets generated during the current study will be available upon reasonable request.
Declarations
Ethics Approval The study design was reviewed and approved by the Careggi University Hospital Ethical Committee (Prot. 2011/0020149 - Rif CEAVC Em. 2019-201 26/11/2019). Ethical approval was waived by the Medical Ethical Committee (MERC) of the University Medical Center Utrecht in view of the retrospective nature of the study. The procedures used in this study adhere to the tenets of the Declara- tion of Helsinki.
Conflict of Interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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