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European Journal of Cancer
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Germline NGS targeted analysis in adult patients with sporadic adrenocortical carcinoma
Maria Scatolini ª,1, Salvatore Grisanti b,1, Pasquale Tomaiuolo a,C, Enrico Grosso ª, Vittoria Basile, Deborah Cosentini b, Soraya Puglisi ”,*, Marta Laganà b, Paola Perotti ”, Laura Saba , Elisa Rossini , Flavia Palermo ª, Sandra Sigala ”, Marco Volante e, Alfredo Berruti b,2, Massimo Terzolo c,2
a Molecular Oncology Laboratory, Fondazione Edo ed Elvo Tempia, 13875 Ponderano, BI, Italy
b Medical Oncology Unit, Department of Medical and Surgical Specialties, Radiological Sciences, and Public Health, University of Brescia, ASST Spedali Civili, 25123 Brescia, Italy
” Internal Medicine, Department of Clinical and Biological Sciences, S. Luigi Gonzaga Hospital, University of Turin, 10043 Orbassano, Italy
d Department of Molecular & Translational Medicine, Section of Pharmacology, University of Brescia, 25123 Brescia, Italy
e Pathology Unit, Oncology department, University of Turin, San Luigi Gonzaga University Hospital, Regione Gonzole 10, 10043 Orbassano, Turin, Italy
ARTICLE INFO
Keywords:
Genetics Pathogenesis
Mutation Oncogenes Tumor suppressors Progression.
Survival Outcome Progression Adrenal cancer
ABSTRACT
Background: Adrenocortical carcinoma (ACC) is a rare cancer that arises sporadically or due to hereditary syn- dromes. Data on germline variants (GVs) in sporadic ACC are limited. Our aim was to characterize GVs of genes potentially related to adrenal diseases in 150 adult patients with sporadic ACC.
Methods: This was a retrospective analysis of stage I-IV ACC patients with sporadic ACC from two reference centers for ACC in Italy. Patients were included in the analysis if they had confirmed diagnosis of ACC, a frozen peripheral blood sample and complete clinical and follow-up data. Next generation sequencing technology was used to analyze the prevalence of GVs in a custom panel of 17 genes belonging to either cancer-predisposition genes or adrenocortical-differentiation genes categories.
Results: We identified 18 GVs based on their frequency, enrichment and predicted functional characteristics. We found six pathogenic (P) or likely pathogenic (LP) variants in ARMC5, CTNNB1, MSH2, PDE11A and TP53 genes; and twelve variants lacking evidence of pathogenicity. New unique P/LP variants were identified in TP53 (p. G105D) and, for the first time, in ARMC5 (p.P731R). The presence of P/LP GVs was associated with reduced survival outcomes and had a significant and independent impact on both progression-free survival and overall survival.
Conclusions: GVs were present in 6.7 % of patients with sporadic ACC, and we identified novel variants of ARMC5 and TP53. These findings may improve understanding of ACC pathogenesis and enable genetic counseling of patients and their families.
1. Introduction
Adrenocortical carcinoma (ACC) is a rare and aggressive neoplasm that arises either sporadically or in the context of hereditary cancer syndromes [1-3]. Studies in southern Brazil, where the incidence of ACC is exceedingly high, have linked ACC development to germline TP53
mutations frequently found in the local population [4,5]. Because of the rarity of ACC, population-based registries of patients with hereditary ACC living in countries other than Brazil are lacking, and current knowledge regarding the heritable fraction of ACC mainly comes from linkage studies of families with hereditary cancer syndromes (Li-Frau- meni syndrome [LFS], Beckwith-Wiedemann syndrome [BWS], Lynch
* Correspondence to: Internal Medicine 1, Department of Clinical and Biological Sciences, San Luigi Gonzaga Hospital, Regione Gonzole 10, 10043 Orbassano, Italy.
E-mail address: soraya.puglisi@unito.it (S. Puglisi).
1 MS and SG contributed equally and should be considered as joint first authors.
2 AB and MT contributed equally and should be considered as joint senior authors.
https://doi.org/10.1016/j.ejca.2024.114088
syndrome, Multiple Endocrine Neoplasia Type 1 [MEN1], and Carney Complex) [1,6,7]. Germline variants (GVs) of specific cancer-associated genes have rarely been assessed in adult ACC patients [1].
The Cancer Genome Atlas (TCGA) project analyzed germline alter- ations related to ACC in two key studies: a pan-cancer study and a specific ACC study [8,9]. Apart from TCGA analysis, most studies have focused only on a limited number of genes [10]. From the analysis of the core dataset of 91 ACC cases in TCGA pan-cancer study [8], a low rate of GVs was found, which places adult sporadic ACC in the lowest quartile among the 33 cancers screened. In the TCGA-ACC study, nine GVs were found among 177 genes potentially linked to ACC [9].
Given the sparse evidence available on GVs in sporadic ACC, the present study aimed to evaluate the frequency and clinical implications of GVs in a targeted group of genes potentially related to adrenal dis- eases in 150 adult patients with ACC. To the best of our knowledge, this study represents the largest multigene germline analysis of adult pa- tients with ACC.
2. Methods
2.1. Study overview
This study was conducted at two reference centers in Italy (San Luigi Hospital, Orbassano, and A.S.S.T. Spedali Civili Hospital, Brescia). Next- Generation Sequencing (NGS) and bioinformatics analyses were con- ducted at the Molecular Oncology Laboratory, Edo, and Elvo Tempia Foundation (Biella). All subjects included in the study provided written informed consent. The study was approved by the Institutional Review Boards of each institution and was conducted in accordance with the Declaration of Helsinki, Good Clinical Practice (GCP), and in compliance with local regulations.
2.2. Patients
A retrospective cohort of patients with ACC was obtained from two institutional review board-approved biological sample repositories, established independently at both centers. Each repository included a collection of peripheral blood samples with comprehensive clinical annotation. Family and clinical histories were obtained through medical documentation and patient interviews with expert medical personnel.
Patients with ACC consecutively referred to our centers between January 1998 and March 2019 were included if they met the following inclusion criteria: age ≥ 18 years, pathologically confirmed diagnosis according to the Weiss criteria [11,12], availability of a peripheral whole blood sample, and complete follow-up information. For oncocytic ACC, Lin-Weiss-Bisceglia classification was used according to the WHO classification, 5th edition [13]. The entire study cohort included 150 patients with presumably sporadic ACC, 32 patients with benign adre- nocortical adenoma, and seven healthy controls. None of our patients had a family history of ACC, or was known to harbor a genomic alter- ation that would increase their risk for ACC, or had clinical character- istics suggestive of genetic syndromes associated with ACC.
2.3. NGS custom panel design
NGS custom panel was designed to cover the coding sequence and flanking region (20 bp) of the following 17 candidate genes: AIP, APC, ARMC5, ARNT, BRCA1, BRCA2, CTNNB1, IGF2, MEN1, MSH2, MSH6, PDE8B, PDE11A, PRKACA, PRKACB, PRKAR1A, and TP53. The panel genes were selected from the recommended list of 56 genes of the American College of Medical Genetics and Genomics (ACMG) for genomic reporting [14] based on literature evidence of their possible role in the pathogenesis of adrenal tumors [1,15-28] (Appendix Table S1). Specifically, six genes (APC, BRCA1, BRCA2, MSH2, MSH6, and TP53) are known to be cancer-predisposing genes according to the ACMG criteria, and 11 genes (AIP, ARNT, ARMC5, CTNNB1, IGF2,
MEN1, PDE8, PDE11A, PRKACA, PRKACB, and PRKAR1A) are involved in pathways linked to adrenal tumorigenesis.
2.4. NGS analysis and bioinformatic interpretation
Germline DNA was isolated from leukocytes in peripheral whole blood samples using standard techniques. NGS analysis was performed using Ion Torrent technologies (Thermo Fisher Scientific) as previously described [29] (see Appendix for details).
A semi-automated bioinformatics pipeline was used, which involved manual inspection of the data quality and contributions from molecular biologists, bioinformaticians, and clinicians. Variant prioritization was calculated after the filtering steps. Polymorphisms in intronic regions or those classified in the ClinVar database [30] as benign or likely benign were excluded. To predict the impact of each amino acid substitution on the structure and function of a protein, each mutation was studied using three in silico tools: Polyphen-2, SIFT, and Grantham [31-33]. In addition, one molecular and one clinical geneticist independently evaluated all variants according to the ACMG rules [32] using literature, public databases, and variant-specific databases (IARC TP53, LOVD, and HGMD). Variants interpreted as pathogenic (P), likely pathogenic (LP), or increased risk alleles were considered as potentially pathogenic [34]. Selected variants were confirmed by Sanger Sequencing in leukocytes and archival FFPE tumor samples, if available (see Appendix for details).
2.5. Statistical analysis
Descriptive statistics were used to analyze the clinical indicators. Associations between variables were assessed using appropriate statis- tical tests. No imputation was performed for missing data.
Overall survival (OS) was calculated from the diagnosis of ACC to death or the date of the last follow-up. Disease-free survival (DFS) was defined as the time from radical surgery to the first radiological evidence of ACC relapse or date of the last follow-up. Progression-free survival (PFS) was calculated from diagnosis to the first evidence of progressive disease (PD) or death in metastatic patients, and from disease relapse to progression, death, or last follow-up in non-metastatic patients.
Survival curves were generated using the Kaplan-Meier method and compared using the log-rank test. Known clinical variables with a po- tential prognostic value for each survival endpoint (enter level p ≤ 0.05, univariate analysis) were included in the multivariate Cox models. Race was not controlled because 98 % of the patients were European non- Finnish. The results were reported as hazard ratios (HR) with 95 % confidence intervals (95 %CI). Cohen’s d value was calculated to mea- sure the effect of germline variant size on survival endpoints. For all tests, statistical significance was set at P < 0.05. All analyses were performed using SPSS v.23.0 (IBM-SPSS Statistics, USA) and R Core Team (2020) version 4.0.2.
3. Results
3.1. Patient characteristics
Our cohort of 150 patients had a median age at diagnosis of 47 years (range 18-82) and male-to-female ratio of 1:1.83. The patient charac- teristics are described in Appendix Table S2.
A personal history of cancer other than ACC was found in 20 patients (13.4 %), and 44 patients (29.4 %) had a family history of cancer. At diagnosis, the majority of ACCs were ENSAT stages I-II (58.7 %). Excess hormones were detected in 55.3 % of cases, and the majority (93.4 %) of patients underwent upfront surgery (see Appendix for details).
3.2. Characterization of germline variants
Of the 150 patients, 21 (14 %) had 18 unique germline variants (GVs) in the panel of analyzed genes (Figure 1). These unique variants were
Mutation
4
2
Mutation by patients
0
3
3
3
2
2
2
1
1
1
2
1
0
Genes
ARMC5
PDE11A
MSH2
TP53
MSH6
PDE8B
CTNNB1
APC
AIP
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
identified in nine different genes: APC (n = 1), ARMC5 (n = 3), MSH2 (n = 3), PDE11A (n = 3), TP53 (n=2), MSH6 (n=2), PDE8B (n=2), AIP (n =1), and CTNNB1 (n =1) (Table 1). The 18 GVs found in our patients were classified using ACMG classification as follows: a) 6 pathogenic (P) or likely pathogenic (LP) variants in ARMC5, CTNNB1, MSH2, PDE11A, and TP53 (n = 2) genes; b) 12 variants in AIP, APC, ARMC5, MSH2, MSH6, PDE11A and PDE8B lacking a clear evidence of pathogenicity. Overall, 10 of the 150 (6.7 %) patients had potentially pathogenic variants.
Specifically, 15 missense mutations and three in-frame deletions were identified (Appendix Table S3). Supplemental Fig. S1 shows the corresponding human proteins with their functional domains and al- terations. Most variants described in this series were present in a single patient at a higher frequency than those reported for GnomAD. The variants p.P731R in ARMC5, p.R307X and p.I552T in PDE11A were particularly enriched, as they were found in more than one patient (Appendix Table S4). Two patients had concurrent mutations involving MSH2 and CTNNB1, PDE11A and PDE8B genes, respectively (Appendix Table S5). The TP53 p.G105D variant was found in a female patient whose sister was later diagnosed with ACC and was confirmed to carry the TP53 p.G105D variant. Sanger sequencing of the specific regions of interest was performed on archival FFPE tumor samples of patient #113, and the TP53 p.R110P mutation was detected in the absence of the gene- negative allele (LOH). In patient #117, Sanger sequencing showed the ARMC5 p.P731R variant with a frequency comparable to germline expression (heterozygous). No other pathogenic variant was found by analyzing the entire coding sequence of ARMC5.
With our experimental approach and bioinformatics pipeline, no
deleterious variants were found in 32 subjects with adrenal adenoma and seven healthy controls.
3.3. Histological analysis of patients with germline variants
Table 2 summarizes the results of the histological analysis of 10 of 21 patients carrying potentially pathogenic GVs. Predominant oncocytic features were observed in the MSH2 mutated patient and immunohis- tochemistry for mismatch repair proteins showed an altered pattern of MSH6 protein. One oncocytic and one conventional ACC showing an altered p53 pattern on immunohistochemistry with protein over- expression were present in patients with TP53 germline variants. PDE11A and ARMC5 mutated patients had ACC with a minor component of the oncocytic or myxoid subtype. One PDE11A-associated case showed a well-circumscribed lesion surrounded by a thin capsule, with a Weiss score of 5 and unequivocal signs of vascular invasion (Supple- mental Fig. S2). One ARMC5-associated case showed the combined features of ACC and macronodular cortical nodular disease (Supple- mental Figure A3).
3.4. Correlation between germline genotype and clinical phenotype
Table 2 reports the clinical characteristics of patients carrying GVs. Women represented 80.0 % of the 10 carriers of potentially pathogenic variants, and their median age at diagnosis was 58.5 years (range, 29-72 years). Demographic and clinical characteristics did not significantly differ between patients who were or were not carrying GVs (Table 3). Despite being diagnosed with apparently sporadic ACC, 40.0 % of
| Germline Variants Identified | ExAC# frequency | dbSNP | ClinVarID | ACMG Classification | Patient ID |
|---|---|---|---|---|---|
| APC (NM_000038.6) | |||||
| c.3410A>G p.(D1137G) | NA | rs1765418674 | 836538 | VUS | #022 |
| ARMC5 (NM_001105247.2) | |||||
| c .26C>T p.(T9M) | NA | rs1166729776 | NA | VUS | #057 |
| c.66_68del p.(A23del) | NA | rs778338263 | NA | VUS | #103 |
| c .2192C>G p.(P731R) | 1.91e-03 | rs200951744 | 1303338 | LP | #089 #107 #117 |
| MSH2 (NM_000251.3) | |||||
| c .136C>G p.(H46D) | NA | rs1553348821 | 1058599 | VUS | #131 |
| c.1786_1788del p.(N596del) | 1.50e-05 | rs63749831 | 1757 | P | #112 |
| c.1804C>G p.(L602V) | 1.50e-05 | rs748797209 | 219668 | VUS | #032 |
| PDE11A (NM_016953.4) | |||||
| c .919C>T p.(R307X) | 4.45e-03 | rs76308115 | 5286 | LP | #011 #063 #109 |
| c .1655T>C p.(I552T) | 1.46e-03 | rs138427178 | 725066 | VUS | #148, #077 |
| c. 2531G>C p.(R844P) | 1.50e-05 | NA | NA | VUS | #088 |
| TP53 (NM_000546.6) | |||||
| c .314G>A p.(G105D) | NA | rs587781504 | 141114 | LP | #102 |
| c .329G>C p.(R110P) | NA | rs11540654 | 233627 | P | #113 |
| MSH6 (NM_000179.3) | |||||
| c .3788G>A p.(R1263H) | 7.50e-05 | rs147852216 | 127593 | VUS | #042 |
| c .3800T> C p.(M1267T) | 1.60e-04 | rs148445930 | 142672 | VUS | #134 |
| PDE8B (NM_003719.5) | |||||
| c .1183C>T p.(R395C) | 4.50e-05 | rs778969486 | NA | VUS | #049 |
| c .1831T>G p.(S611A) | 1.50e-05 | rs201596222 | 906328 | VUS | #109 |
| AIP (NM_003977.4) | |||||
| c .161G>A p.(R54Q) | 3.01e-05 | rs762938281 | 819687 | VUS | #017 |
| CTNNB1 (NM_001904.4) | |||||
| c.2262_2300del | NA | NA | NA | LP | #032 |
| p.(D755_P767del) |
Legend of abbreviations in alphabetical order. LP, likely pathogenic; P, pathogenic; VUS, variant of uncertain significance; # European (non-Finnish), ExAC v1.0.
patients with GVs had a family history of cancer (with a median of three relatives) compared to 28.6 % of patients who did not. Moreover, one patient with the TP53 p.G105D variant had a previously unknown family history of ACC. The frequency of a personal history of cancer did not differ between patients who carried GVs and those who did not (Table 3).
3.5. Survival outcomes and prognostic factors
The database was closed for the final analysis on October 20, 2022. At that time, 94 of the 127 (74.0 %) operated patients had disease relapse and 100 % (23/23) of the metastatic patients had disease pro- gression. Overall, 82 patients (54.6 %) were alive and 50/150 (33.3 %) were alive and free from progression. The median OS in the entire series was 142 months (range, 1-297 months) and the 5 year-OS was 65 %. The median DFS and PFS were 31 months (range, 1-225+ months) and 27 months (range, 1-275+ months), respectively (Fig. 2).
Univariate and multivariate analyses of clinical characteristics showed that ENSAT stage and resection of the primary tumor were prognostic factors for both OS and PFS, whereas age and cortisol excess significantly affected OS. Adjuvant treatment and a familial history of cancer were prognostic factors for DFS (Appendix Table S6). In uni- variate analysis, potentially pathogenic GVs were significant predictors of PFS (HR 2.39, 95 %CI, 1.09-5.27; p = 0.029) and OS (HR 2.18, 95 % CI, 1.01-4.80; p = 0.046) (Appendix Table S7-S8). The multivariate Cox model retained the prognostic significance of potentially pathogenic GVs for both endpoints (PFS, HR 2.41, 95 %CI, 1.05-5.53; p = 0.037; OS, HR 2.43, 95 %CI, 1.01-5.84; p = 0.046) (Appendix Table S7-S8). Among patients with metastatic disease, carriers of potentially patho- genic GVs had a median PFS of 9 months versus 27 months in gene- negative patients (p = 0.023). The corresponding median OSs were 39 and 142 months (p = 0.046), respectively (Fig. 2).
4. Discussion
We investigated the presence of GVs in 17 selected genes (NGS
custom panel) in 150 adult patients with sporadic ACC. We investigated known cancer-predisposing genes (APC, BRCA1, BRCA2, MEN1, MSH2, MSH6, and TP53) linked to ACC, mostly in the context of hereditary syndromes [1] because limited knowledge is available on whether they are associated with sporadic ACC [35]. We also included genes (AIP, ARMC5, ARNT, CTNNB1, IGF2, PDE8B, PDE11A, PRKACA, PRKACB, PRKAR1A) involved in several endocrine diseases characterized by disruption of specific adrenal cortex pathways, or affected by somatic mutations in ACC [9,36-38].
By applying this “plausible association” approach, we found that 21 (14 %) of 150 adult patients with apparently sporadic ACC were carriers of at least one GV, and 10 (6.7 %) carried potentially pathogenic vari- ants. This frequency of GV carriers is similar to that reported in an ACC- specific TCGA study [9].
The prevalence of GVs in adult patients was lower than that previ- ously reported in pediatric patients [39], thus supporting the hypothesis that the prevalence of germline variants is inversely correlated with age [40]. In our series, 40 % of patients with potential pathogenic GVs had a family history of cancer, an intriguingly high figure that may suggest a genetic predisposition in some of these cases. Our results underscore the need to offer patients with presumed sporadic ACC genetic counseling to identify an underlying hereditary syndrome [1].
Most of the variants described in our series were present in indi- vidual patients, and some variants had a frequency that was much higher than that reported in the GnomAD database. This enrichment suggests a new, yet not described, role for these variants in the patho- genesis of ACC.
However, some patients are carriers of multiple variants. In fact, we identified the co-presence of variants in MSH2 and CTNN1B, PDE11A and PDE8B. Patients who carried multiple GVs did not appear to have a worse prognostic profile than those who did not, and their baseline characteristics did not differ significantly from those of the others.
We identified one potentially pathogenic variant in MSH2. This finding supports the view that ACC may be considered a part of Lynch syndrome [7,21] and highlights the role of the GVs of genes involved in DNA damage repair in the pathogenesis of ACC. However, we have to
| Patient ID | Gender | Age at diagnosis (years) | OS (months) | Live status | Personal history of cancer other | Family history of cancer | ACC stage | Secretion of ACC | Gene/Variant | ACMG classification | Complementary IHC | Histological variant | Weiss score | Helsinki score | Ki- 67 (%) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| than ACC | |||||||||||||||
| #089 | F | 60 | 44 | DOD | NO | NO | II | Cosecretion of | ARMC5 p. | LP | Not executable | Focal | 6 | 18 | 10 |
| cortisol and | P731R | oncocytic (20 | |||||||||||||
| other steroids | %) | ||||||||||||||
| #107 | M | 57 | 55 | ANED | NO | NSCLC, CRC, | II | Cosecretion of cortisol and | ARMC5 p. P731R | LP | NA | NA | NA | NA | NA |
| BLCA, PCa | other steroids | ||||||||||||||
| #117 | M | 64 | 74 | DOD | NO | NO | I | Cosecretion of cortisol and | ARMC5 p. P731R | LP | No evaluable target | Focal myxoid (10 %) | 4 | 23 | 20 |
| other steroids | |||||||||||||||
| #032 | F | 72 | 36 | DOD | NO | NO | I | Cosecretion of cortisol and | CTNN1B p. D755_767del | LP | NA | NA | NA | NA | NA |
| other steroids | |||||||||||||||
| #112 | F | 65 | 2 | DOD | NO | CRC | IV | No secretion | MSH2 p. N596del | P | MSH6 negative (altered pattern) | Oncocytic variant | NA (biopsy) | NA (biopsy) | 20 |
| #011 | F | 29 | 76 | ANED | NO | NO | I | No secretion | PDE11A p. R307X | LP | Not executable | Focal oncocytic (30 %) | 5 | 3 | 3 |
| #063 | F | 36 | 27 | DOD | NO | CRC, NSCLC | III | No secretion | PDE11A p. R307X | LP | Not executable | Myxoid(40 %) | 9 | 78 | 70 |
| #109 | F | 42 | 92 | ANED | NO | NO | II | No secretion | PDE11A p. R307X | LP | No evaluable target | Focal oncocytic (30 %) | 5 | NA | NA |
| #102 | F | 35 | 39 | DOD | NO | ACC, NSCLC, | II | Cosecretion of cortisol and | TP53 p.G105D | LP | Not executable | Oncocytic variant | NA | 23 | 18 |
| LGG, | other steroids | ||||||||||||||
| others | |||||||||||||||
| #113 | F | 61 | 34 | DOD | BRCA | NO | III | No secretion | TP53 p.R110P | P | p53 overexpressed (altered pattern) | Conventional | 7 | 48 | 40 |
Legend of abbreviations in alphabetical order. ACC, adrenocortical carcinoma; ANED, alive with no evident disease; BLCA, bladder carcinoma; BRCA, breast carcinoma; CRC, colorectal cancer; DOD, dead of disease; LGG, low-grade glioma; LP, likely pathogenic; NA: not available; NSCLC, non-small cell lung cancer; P, Pathogenic; PCa, prostate cancer.
| Variable | Patients with potentially pathogenic GVs (n =10) | Patients without potentially pathogenic GVs | p- value | |
|---|---|---|---|---|
| (n =140) | ||||
| Age, (years) | Median | 58.5 | 47.0 | 0.268 |
| Range, (IQR) | 29-72 (28.5) | 18-82 (21.7) | ||
| Sex, n (%) | M | 2 (20) | 51 (36) | 0.479 |
| F | 8 (80) | 89 (64) | ||
| ENSAT stage, n (%) | Stage I-II | 7 (70) | 81 (58) | 0.748 |
| Stage III | 2 (20) | 37 (26) | ||
| Stage IV | 1 (10) | 22 (16) | ||
| Clinical | No | 5 (50) | 62 (44) | 0.982 |
| hypersecretion, n (%) | Yes | 5 (50) | 78 (56) | |
| Type of | Hypercortisolism | 4 (40) | 50 (36) | 0.747 |
| hypersecretion, n (%) | Other steroids | 6 (60) | 90 (64) | |
| Proliferation | Median | 20ª | 20b | 0.778 |
| index (KI67 %) | Range, (IQR) | 4-35 (12.5) | 2-75 (20) | |
| Tumor size, (cm) | Median | 7.5 | 10℃ | 0.254 |
| Range, (IQR) | 4-21 (10) | 2-25 (3.5) | ||
| Weiss score | Median | 7.5ª | 6e | 0.084 |
| Range, (IQR) | 4-9 (3) | 2-9 (6.3) | ||
| Surgery of primary tumor, n (%) | No | 1 (10) | 9 (6) | 0.510 |
| Yes | 9 (90) | 131 (94) | ||
| Adjuvant | No | 3 (30) | 46 (33) | 1.000 |
| treatment, n (%) | Yes | 7 (70) | 94 (67) | |
| Personal history | No | 9 (90) | 121 (86) | 1.000 |
| of cancer other than ACC, n (%) | Yes | 1 (10) | 19 (14) | |
| Family history of | No | 6 (60) | 100 (71) | 0.480 |
| cancer, n (%) | Yes | 4 (40) | 40 (29) |
Mann Whitney was used for continuous variables, Fisher exact test for ordinary variables.
Legend: ª available on 7 patients; b available on 129 patients; ” available on 129 patients; ª available on 6 patients; e available on 113 patients.
acknowledge that our gene panel did not cover the whole genomic spectrum of Lynch syndrome. Moreover, a partially discordant result was observed between genomic profiling and immunohistochemistry for MMR proteins, since MSH2 protein expression was preserved. However, MSH2 protein retained expression has been described in patients harboring germline MSH2 variants [41], possibly as the consequence of an alteration of the protein function but not of the expression of the protein domain that acts as epitope for the antibody. Indeed, the loss of MSH6 protein in this patient further support a damage of the MSH2/MSH6 complex and the pathogenicity of the MSH2 variant detected.
Of the two GVs detected in TP53, the p.G105D variant has been observed at low frequencies in large population studies [42]. This variant produces an in-frame deletion at the end of exon 4, as observed in patients with breast cancer [43]. We found this variant in a 35-year — old female and subsequently in her sister, who was found to have androgen-secreting ACC at the age of two years. The fact that the p. G105D variant was found in two patients from the same family suggests a pathogenic role for this variant, which fits well with its localization in a highly conserved protein domain. According to the Chompret criteria that have been recently proposed to identify affected families beyond the classical criteria of Li-Fraumeni syndrome [44], this is a Li-Fraumeni family that was previously unknown and recognized through the study.
The p.R110P variant has previously been reported in two males from separate families with Li-Fraumeni syndrome, one with gastric cancer at 32 years of age and the other with two primary sarcomas at 37 and 44
years of age [45,46] as well as in an individual with soft tissue sarcomas [47]. Several functional studies have demonstrated that this alteration is deficient in transcriptional activation, DNA binding, apoptosis induc- tion, and cell growth suppression [48,49]. One study suggested the as- sembly of mutant p53 into large aggregates, resulting in impaired nuclear import [50]. We identified this variant in a 61-year-old female with ACC and breast cancer. Immunohistochemical analysis confirmed cytoplasmic overexpression of TP53 protein in the tumor, and Sanger sequencing showed the presence of LOH for the variant in the TP53 allele. Neither of these TP53 variants has previously been reported in patients with ACC, and our findings suggest that they may play a role in ACC development.
ARMC5 is a tumor suppressor gene responsible for the familial form of primary bilateral macronodular adrenal hyperplasia (PBMAH). The presence of inactivating ARMC5 mutations is associated with a severe form of ACTH-independent Cushing syndrome as well as an overall in- crease in adrenal mass [23,51,52]. For these reasons, it has been sug- gested (but never confirmed) that the GVs of ARMC5 represent a genetic risk factor for ACC [38,53].
We detected one variant of ARMC5 in three patients (0.2 %), which has already been reported in PBMAH [23]. We identified the p.P731R variant in three patients whose clinical characteristics included older age, large tumors, and cortisol excess. Such findings reflect those observed in patients with PBMAH [23,38]. In contrast to the findings in PBMAH [38], we were unable to demonstrate a secondary alteration of the ARMC5 gene in one patient with available tumor material. To the best of our knowledge, this is the first report of GVs in the ARMC5 gene in patients with ACC. Interestingly, one patient carrying the ARMC5 variant displayed pathological features of both ACC and PBMAH. This finding leads to the hypothesis that progression from benign to malig- nant cortical proliferation, from PBMAH to ACC, is possible.
Recent evidence supports the notion that germline mutations may contribute to tumor progression [54,55]. In our series, potentially pathogenic GVs were associated with reduced survival outcomes and had a significant and independent impact on both PFS and OS.
This study had some limitations. First, we focused on a set of genes that did not encompass the full genetic variability of ACC: due to shortage of funding, we did not perform whole exome sequencing. Therefore, the frequency of pathogenic GVs may have been under- estimated in this study. Second, we were unable to perform a systematic parallel analysis of somatic DNA since most patients were referred to us after being operated in other centers. Thus, we could not thoroughly investigate the double-hit events and clearly ascertain the specific pathogenicity of individual variants.
In conclusion, we have characterized the largest series to date of adult patients with sporadic ACC for GVs using an NGS target gene panel. Our results showed a 6.7 % prevalence of potentially pathogenic variants, which were mainly found in genes involved in DNA damage repair. In addition, we reported, for what we believe is the first time, the presence of GVs of ARMC5 in patients with ACC, and we found two novel pathogenic variants of TP53. The present study thus extends the knowledge on the germline component in this rare cancer and highlights the role of genetic counseling for patients with apparently sporadic ACC and their families.
CRediT authorship contribution statement
Deborah Cosentini: Writing - review & editing, Visualization, Validation, Data curation, Investigation. Soraya Puglisi: Writing - re- view & editing, Visualization, Validation, Investigation, Data curation. Marta Laganà: Writing - review & editing, Visualization, Validation, Investigation, Data curation. Paola Perotti: Writing - review & editing, Visualization, Validation, Project administration. Laura Saba: Writing - review & editing, Visualization, Validation, Investigation, Data cura- tion. Elisa Rossini: Writing - review & editing, Visualization, Valida- tion, Investigation, Data curation. Sandra Sigala: Writing - review &
1,0
Median DFS
27 vs 32 months
1,0
Median OS Log-rank test
39 vs 142 months
w/o PP GVs
Log-rank test
p 0.529
p 0.046
with PP GVs
Univariate HR
1.27 95%CI 0.59-2.77
Univariate HR
2.18 95%CI 0.99-4.80
censored
censored
0,8-
0,8-
Disease-free survival (%)
A
Overall survival (%)
B
0,6-
0,6-
0,4
0,4-
0,2
0,2
0,0
0,0
0
50
100
150
200
250
0
50
100
150
200
250
300
Time (months)
Time (months)
No. at risk
No. at risk
118
41
18
4
1
0
140
92
40
15
5
3
0
9
3
1
0
0
0
10
4
1
0
0
0
0
1,0
Median PFS
9 vs 27 months
1,0
Median OS M+ pts Log-rank test
36 vs 80 months
Log-rank test
p 0.023
p 0.076
w/o PP GVs
Univariate HR
2.39 95%CI 1.09-5.27
Univariate HR
1.81 95%CI 0.86-3.82
with PP GVs
censored
censored
Progression-free survival (%)
0,8-
0,8-
C
Overall survival (%)
D
0,6-
0,6-
0,4
0,4
0,2-
0,2
0,0-
0,0
0
50
100
150
200
250
300
0
50
100
150
200
250
300
Time (months)
No. at risk
No. at risk
Time (months)
109
31
10
5
3
1
0
109
66
28
12
6
3
0
8
0
0
0
0
0
0
8
2
0
0
0
0
0
editing, Visualization, Validation, Supervision. Maria Scatolini: Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Methodology, Formal analysis, Conceptualiza- tion. Salvatore Grisanti: Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Methodology, Formal analysis, Conceptualization. Marco Volante: Writing - review & edit- ing, Visualization, Validation, Supervision. Alfredo Berruti: Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Conceptualization. Pasquale Tomaiuolo: Writing - review & editing, Writing - original draft, Visualization, Validation, Software, Investigation. Flavia Palermo: Validation, Visualization, Writing -
review & editing. Massimo Terzolo: Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Re- sources, Funding acquisition, Conceptualization. Enrico Grosso: Writing - review & editing, Visualization, Validation, Software, Inves- tigation. Vittoria Basile: Writing - review & editing, Visualization, Validation, Investigation, Data curation.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
We thank all the patients for their participation in this study.
Financial support
This work was funded by the Associazione Italiana per la Ricerca sul Cancro (AIRC), grant number IG2019-23069 to Massimo Terzolo.
Conflict of interest
The authors declare no potential conflicts of interest.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ejca.2024.114088.
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