Accepted Manuscript
Sarcomatoid Adrenocortical Carcinoma: A Comprehensive Pathological, Immunohistochemical and Targeted Next-Generation Sequencing Analysis
Human PATHOLOGY
W: B. Saunders
Thomas G. Papathomas MD, PHD, Eleonora Duregon MD, Esther Korper- shoek PhD, David F. Restuccia PhD, Ronald van Marion, Rocco Cappellesso MD, Nathalie Sturm MD, PhD, Giulio Rossi MD, PhD, Antonella Coli MD, Nicola Zucchini MD, Hans Stoop PhD, Wolter Oosterhuis MD, PhD, Laura Ventura PhD, Marco Volante MD, PhD, Ambrogio Fassina MD, PhD, Winand N.M. Dinjens PhD, Mauro Papotti MD, PHD, Ronald R. de Krijger MD, PhD
| PII: | S0046-8177(16)30204-0 |
| DOI: | doi: 10.1016/j.humpath.2016.08.006 |
| Reference: | YHUPA 3991 |
| To appear in: | Human Pathology |
| Received date: | 7 April 2016 |
| Revised date: | 3 August 2016 |
| Accepted date: | 18 August 2016 |
Please cite this article as: Papathomas Thomas G., Duregon Eleonora, Korpershoek Esther, Restuccia David F., van Marion Ronald, Cappellesso Rocco, Sturm Nathalie, Rossi Giulio, Coli Antonella, Zucchini Nicola, Stoop Hans, Oosterhuis Wolter, Ventura Laura, Volante Marco, Fassina Ambrogio, Dinjens Winand N.M., Papotti Mauro, de Krijger Ronald R., Sarcomatoid Adrenocortical Carcinoma: A Comprehensive Patholog- ical, Immunohistochemical and Targeted Next-Generation Sequencing Analysis, Human Pathology (2016), doi: 10.1016/j.humpath.2016.08.006
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Title Page
Sarcomatoid Adrenocortical Carcinoma: A Comprehensive Pathological, Immunohistochemical and Targeted Next-Generation Sequencing Analysis.
Thomas G. Papathomas MD, PHD 1,2 *, Eleonora Duregon MD 3*, Esther Korpershoek PhD 4, David F. Restuccia PhD 5, Ronald van Marion 6, Rocco Cappellesso MD 7, Nathalie Sturm MD, PHD 8, Giulio Rossi MD, PHD 9, Antonella Coli MD 10, Nicola Zucchini MD 11, Hans Stoop PhD 12, Wolter Oosterhuis MD, PhD 13, Laura Ventura PhD14, Marco Volante MD, PHD 15, Ambrogio Fassina MD, PhD 16, Winand N.M. Dinjens PhD 17, Mauro Papotti MD, PhD 18 *, Ronald R. de Krijger MD, PHD 19, 20 *
* equal contribution
1. Department of Pathology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam 3015 GE, The Netherlands. Electronic address: t.papathomas@erasmusmc.nl
2. Department of Histopathology, King’s College Hospital, Denmark Hill, London SE5 9RS, UK. Electronic address: thomaspapathomas@nhs.net
3. Department of Oncology, University of Turin, Orbassano 10043, Turin, Italy. Electronic address: eleonora.duregon@unito.it
4. Department of Pathology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam 3015 GE, The Netherlands. Electronic address: e.korpershoek.1@erasmusmc.nl
5. Department of Pathology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam 3015 GE, The Netherlands. Electronic address: restuccia.david@gmail.com
6. Department of Pathology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam 3015 GE, The Netherlands. Electronic address: r.vanmarion@erasmusmc.nl
7. Department of Diagnostic Medical Sciences and Special Therapies, Surgical Pathology and Cytopathology Unit, University of Padova, Padova 35121, Italy. Electronic address: rocco.cappellesso@gmail.com
8. Department of Pathology, CHU Albert Michallon, Grenoble 38043, France. Electronic address: NSturm@chu-grenoble.fr
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9. Division of Pathology, University of Modena, Modena 41100, Italy. Electronic address: rossi.giulio@policlinico.mo.it
10. Division of Pathology, Policlinico Universitario Gemelli, Rome 00168, Italy. Electronic address: antonella.coli@rm.unicatt.it
11. Division of Pathology, San Gerardo Hospital, Monza 20900, Italy. Electronic address: n.zucchini@hsgerardo.org
12. Department of Pathology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam 3015 GE, The Netherlands. Electronic address: j.stoop@erasmusmc.nl
13. Department of Pathology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam 3015 GE, The Netherlands. Electronic address: j.w.oosterhuis@erasmusmc.nl
14. Department of Statistical Sciences, University of Padova, Padova 35121, Italy. Electronic address: laura.ventura@unipd.it
15. Department of Oncology, University of Turin, Orbassano 10043, Turin, Italy. Electronic address: marco.volante@unito.it
16. Department of Diagnostic Medical Sciences and Special Therapies, Surgical Pathology and Cytopathology Unit, University of Padova, Padova 35121, Italy. Electronic address: ambrogio.fassina@unipd.it
17. Department of Pathology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam 3015 GE, The Netherlands. Electronic address: w.dinjens@erasmusmc.nl
18. Department of Oncology, University of Turin, Orbassano 10043, Turin, Italy. Electronic address: mauro.papotti@unito.it
19. Department of Pathology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam 3015 GE, The Netherlands. Electronic address: r.dekrijger@erasmusmc.nl
20. Department of Pathology, Reinier de Graaf Hospital, Delft 2625 AD, The Netherlands. Electronic address: r.dekrijger@erasmusmc.nl
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Contact details of the corresponding author:
Thomas G. Papathomas, MD Department of Histopathology, King’s College Hospital, Denmark Hill, London SE5 9RS, UK Tel: +44-7437416650 thomaspapathomas@nhs.net
CRIPT
Acknowledgements: Funding: This study was supported by the Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 259735 (ENS@T-Cancer) and partially supported by a grant from the Italian Association for Cancer Research (AIRC, Milan; grant no. IG/14820/2013 to M.P.). D.F.R. was supported by a ParaPheoAlliance Fellowship. ☒ 4/
Conflict of Interest/ Disclosure: The authors declare no conflict of interest.
Running Head: A Comprehensive Analysis of Sarcomatoid Adrenocortical Carcinoma
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Abstract
Adrenocortical carcinomas (ACCs) with sarcomatous areas represent an extremely rare type of highly aggressive malignancy of unknown molecular pathogenesis. The current study was planned to gain insight into its molecular genetics using a targeted next-generation sequencing approach and to explore the status of epithelial-mesenchymal transition (EMT)-associated markers (E-/P-/N-Cadherins, MMP-2/-9 and Caveolin-1), downstream transcriptional regulators of EMT-related signaling pathways (ZEB-1/-2, Slug), stem cell factors (Oct3/4, LIN28, SOX2, SO17, NANOG, CD133, nestin) and markers of adrenocortical origin/ tumorigenesis (SF-1, ß-catenin, p53) in phenotypically diverse tumor components of six cases. Thirteen pathogenic variants of ACC-associated TP53 and CTNNB1 genes were detected in epithelial and/or non-epithelial components in 4 out of 6 tumors. Three cases had identical mutations in distinct components, one of which containing TP53/CTNNB1 in 3 out of 5 components, while one harbored a single TP53 mutation only in the non-epithelial component. By immunohistochemistry, SF-1 and E-/P-/N-Cadherins were found positive only in the epithelial component of all cases, whereas the non-epithelial components were mainly enriched for nestin, ZEB-1 and MMP-2/-9. @-catenin demonstrated an aberrant nuclear localization in the sarcomatoid component of 5 cases, while p53 was strongly positive in non-epithelial constituent in 4 of 6 cases. In summary, we have shown that Wnt/B- catenin signaling pathway dysregulation and mutational inactivation of TP53 are common genetic events in sarcomatoid ACCs, a subset of which being monoclonal in origin. These tumors are enriched for EMT- related markers and stem cell factors, potentially conferring a poor prognosis, which might be exploited as novel therapeutic targets.
Keywords: adrenocortical carcinoma; sarcomatoid; mutations; epithelial-mesenchymal transition; nestin
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Introduction
Adrenocortical carcinomas (ACCs) with sarcomatous areas represent an extremely rare type of malignancy being the least common ACC variant following oncocytic and myxoid ACCs in decreasing order of frequency [1]. As a matter of fact, only 16 cases have been previously published, all of which as single case reports [2-17]. ACCs with sarcomatous areas have been variably designated carcinosarcomas or sarcomatoid carcinomas due to the presence of a specialized mesenchymal component, including osteosarcomatous, chondrosarcomatous or rhabdomyosarcomatous differentiation, or devoid of specific differentiation, respectively [2-17]. Nonetheless, this variant is characterized by a highly aggressive biologic behaviour and a worse prognosis in comparison with its conventional counterparts [1]. The question whether this aggressive clinical course may be attributed not only to advanced stage at presentation, but also to inherent biologic properties, still remains unanswered.
Epithelial-mesenchymal transition (EMT) is a key developmental program that can be aberrantly activated during tumor progression, endowing cells with invasive and migratory properties, inducing stem cell properties, preventing apoptosis and senescence and contributing to chemo-resistance and immunosuppression [18]. During tumor progression, epithelial cells are driven towards a mesenchymal state, which subsequently enables them to evade hostile microenvironments generated by hypoxia, mechanical constraints and/or nutrient deprivation [19]. In this context, it has been shown that the induction of EMT endows breast epithelial cells with stem cell traits [20], while these EMT-derived cells share similarities with mesenchymal stem cells in terms of gene expression, multilineage differentiation, and ability to migrate towards tumor cells and wound sites [21]. Despite these lines of experimental evidence implying a link between EMT and cancer stem cells (CSCs), it still remains unsettled whether CSCs derive from EMT-induced differentiated cancer cells or from transformed tissue-specific stem cells [22]. Immunohistochemical expression of EMT markers has been documented in sarcomatoid carcinomas/carcinosarcomas [23-25], implying a potential role for EMT in such tumors.
The purpose of the current study was to gain insight into the molecular genetics of this rare ACC variant by using a targeted next-generation sequencing (NGS) approach. In addition, we investigated the expression of EMT-associated markers, downstream transcriptional regulators of EMT-related signaling pathways and stem cell factors in morphologically diverse tumor components of sarcomatoid ACCs.
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Materials & Methods
Case series
Six sarcomatoid ACCs were collected from five Departments of Pathology, three of which had previously been reported from the clinico-pathological viewpoint [8-9, 11]. Detailed clinico-pathological data of all cases included in the current study are summarized in Table 1. All cases were reviewed by two investigators (TP/ED) to identify the sarcomatoid areas. The study received ethical approval from the local Institutional Review Boards.
Tissue preparation and immunohistochemistry (IHC)
Sections serial to those used for conventional hematoxylin-eosin were obtained from one or two representative formalin-fixed paraffin-embedded (FFPE) blocks. The following markers were evaluated: (i) general ones of adrenocortical origin and differential diagnosis: pan-cytokeratin AE1/AE3, vimentin, Steroidogenic Factor 1 (SF-1), melan A, a-inhibin, synaptophysin, chromogranin A, neuron specific enolase (NSE), CD56 & Ki-67; (ii) markers involved in ACC tumorigenesis: ß-catenin & p53; (iii) EMT- related markers: E-Cadherin, P-Cadherin, N-Cadherin, Matrix-Metalloproteinases (MMP)-2, MMP-9 & Caveolin-1; (iv) downstream transcriptional regulators of EMT-related signaling pathways: ZEB1, ZEB2 & Slug; (v) stem cell factors: Oct3/4, LIN28, NANOG, SOX2, SOX17, CD133 & nestin. Antibodies ☒ employed, experimental conditions, staining interpretation and scoring systems are detailed in Supplementary Table 1.
Adrenal cortical tumor series
To investigate the specificity of those immunohistochemical markers expressed in sarcomatoid ACCs, i.e. EMT-related markers (N-Cadherin, P-Cadherin, E-Cadherin, MMP2, MMP9 and Caveolin-1), downstream transcriptional regulators of EMT-related signaling pathways (ZEB1) and stem cell factors (nestin, SOX2, SOX17 and LIN28), these were also assessed in 38 conventional ACCs and 17 adrenocortical adenomas (ACAs) (including 12 control tissue samples) arranged in a tissue microarray (TMA) format using an automated TMA constructor (ATA-27 Beecher Instruments, Sun Prairie, WI, USA; available at the Department of Pathology, Erasmus MC Cancer Institute) as previously described [26].
DNA Isolation
From six sarcomatoid ACCs, 16 morphologically distinct tumour components (8 epithelial/ 8 sarcomatoid) were identified, as follows: 1 epithelial/1 sarcomatoid (cases No 1-2/4/6); 2 epithelial/ 3 sarcomatoid (case No 5); and 2 epithelial/ 1 sarcomatoid (case No 3). DNA isolation from 13 tumor areas (cases No 1-2/4-6) was carried out using standard procedures following manual microdissection. DNA isolation from the remaining 3 tumor areas (case No 3) following laser capture microdissection (Zeiss PALM Microbeam IV;
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available at the Department of Pathology, Erasmus MC Cancer Institute). All tumor samples were estimated to contain at least 80% neoplastic cells.
Targeted Next-Generation Sequencing (NGS) analysis
Based on (i) mutations in ACCs, carcinosarcomas occurring at various anatomic locations, anaplastic thyroid carcinomas, and sarcomas; (ii) EMT-related pathways: Wnt pathway, MAPK pathway and PI3K/AKT pathway; and (iii) signalling pathways involved in the pathogenesis of adrenocortical tumors i.e. CAMP/PKA pathway, 18 genes were selected for mutational analysis: (1) APC (exons 12-14), (2) AXIN1 (exons 1-6), (3) AXIN2 (exon 7), (4) CTNNB1 (exon 3), (5) BRAF (exons 11/15), (6) KRAS (exons 2-4), (7) NRAS (exons 2-4), (8) HRAS (exons 2-4), (9) EGFR (exons 18-21), (10) PI3KCA (exons 9/20), (11) AKT1 (exon 2), (12) AKT2 (exon 2), (13) AKT3 (exon 2), (14) PTEN (exons 3-5/7), (15) ALK (exons 23- 25), (16) ERBB2 (exons 19-20), (17) PRKAR1a (exons 4-8) and (18) TP53 (exons 2-11).
An lon AmpliSeq Custom Panel was created and sequenced on the lon Torrent Personal Genome Machine (PGM; Life Technologies) on 10 nanograms (per sample) of FFPE tumor DNA according to the manufacturer’s protocols. In short, libraries were made using the lon AmpliSeq Library Preparation Kit. Template was prepared using the lon OneTouch Template Kit and sequencing was performed with the lon Sequencing Kit v2.0 on an lon 318 chip.
Data were analyzed with Torrent Suite Software, version 3.6 (Life Technologies). Annotation of variant calls was performed with Annovar (http://www.openbioinformatics.org/annovar/) [27] and facilitated using an in-house galaxy platform/server on which Annovar wrapper was installed [28-30]. The variants (i) with a read frequency higher than 30%, (ii) not known as common polymorphisms according to 1000G2012 April and ESP6500, (iii) non-synonymous with a minimum of 5 forward/ reverse variant read and 100 total depth read were retained as variants (mutations) and confirmed by alternate platforms i.e. Sanger direct sequencing. Sequences of all primers and probes are available upon request.
TERT promoter mutation analysis
In two cases displaying identical mutations in morphologically diverse components, hotspot TERT promoter mutations were analyzed by a SNAPshot assay using the ABI Prism SNAPshot Multiplex Kit (Applied Biosystems, Foster City, CA, USA) as previously described [26].
Statistical analysis
Non-parametric tests (Wilcoxon rank sum test) were used to study differences of immunohistochemical expression between the sarcomatoid series and control ACAs and conventional ACCs. Reciprocal correlations among the different expression of immunohistochemical markers among control ACCs and ACAs were made using a two-tailed Spearman’s test.
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Results
Clinical and pathological features of sarcomatoid ACCs
Three unpublished cases analyzed herein further added to the existing body of the literature; thus accounting for 19 cases (Supplementary Table 2). Sarcomatoid ACCs are rare tumors characterized by a wide age range (23-79 years; median age 55; mean age 53.89) and no gender or laterality predilection. These are usually non-functioning tumors of large size (average 14cm; range 6.5cm up to 24cm). Microscopically, they predominantly display a pure spindle cell component (n=11), which can be occasionally admixed with foci of rhabdomyosarcomatous or osteosarcomatous differentiation or undifferentiated PNET-like areas (n=3). Less frequently a pure specialized mesenchymal component i.e. rhabdomyosarcomatous (n=3), osteosarcomatous (n=1) and osteosarcomatous/chondrosarcomatous constituents (n=1), can be detected. These are highly aggressive neoplasms with a high propensity for metastatic and/or recurrent disease and an average post-operative survival of 6.9 months. Two cases developed a second primary malignancy and associated metastatic disease.
Immunohistochemical profile of sarcomatoid ACCs
SF-1 was the sole adrenocortical marker positive in the epithelial component of all cases, but always negative in sarcomatoid areas. Cytokeratin AE1/AE3 was negative in all cases except for a focal positivity in the epithelial component of case No 1 and the sarcomatoid part of cases No 3 and 6. Conversely, vimentin was positive in both components of all cases, except for the epithelial component of case No 2. Neuron specific enolase had always a positive reactivity, while synaptophysin was positive in the epithelial component of four cases. Ki-67 proliferation index as estimated in the epithelial areas had a median value of 28% (range 1%-54%). ß-catenin showed an aberrant nuclear localization in the sarcomatoid component of all but one case (No 2). ß-catenin and p53 immunohistochemical data are summarized in Table 2 (Figure 1).
Expression and/or specificity of EMT-related markers, downstream transcriptional regulators of EMT-related signaling pathways and stem cell factors in sarcomatoid ACCs
Cadherins were positive only in the epithelial component. In particular, E- and P-cadherins were always negative except for case No 2; N-cadherin was negative in all components of cases No 1-2 and positive only in epithelial components of the other cases. Conversely, MMP-2 and/or MMP-9 were positive in sarcomatoid areas of all cases. ZEB-1 immunoreactivity/ ZEB-2 immunonegativity were noted in both components of all cases. Slug and Caveolin-1 were positive in the sarcomatoid component of five and four cases, respectively. Among stem cell markers, nestin was positive in sarcomatoid areas of all cases, while SOX-2, SOX-17 and LIN28 were positive in the sarcomatoid component of four, one and three cases, respectively. OCT3/4, NANOG and CD133 were always negative (Table 3).
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Comparing the immunohistochemical expression of those markers demonstrated in sarcomatoid areas of sarcomatoid ACCs with a control series of conventional ACCs, MMP-2 and MMP-9 were found significantly more often expressed in sarcomatoid areas (Wilcoxon rank test, p<0.0001 and p=0.0002 respectively), while ZEB1 was always positive in sarcomatoid components and mostly negative in conventional ACCs (Wilcoxon rank test, p=0.003). ACAs had a significant positive expression of N- cadherin and P-cadherin as compared to ACCs (p<0.0001), while ZEB-1 and Caveolin-1 were significantly expressed in ACCs rather than in ACAs (p<0.0001). ACA stained mostly positive for N- cadherin and P-cadherin (p<0.0001 for both markers). ZEB1 was always absent in ACA (p=0.005), while Caveolin 1 was positive in the majority of ACC (p<0.0001).
Mutational analysis of sarcomatoid ACCs
Targeted NGS revealed 13 pathogenic, nonsynonymous, variants of ACC-associated genes in epithelial and/or non-epithelial components in 4 out of 6 tumors. Three cases had identical mutations in phenotypically diverse components, one of which containing TP53/CTNNB1 in 3 out of 5 components, whereas one case harbored a single TP53 mutation in the non-epithelial component (Table 2). The epithelial and sarcomatoid tumor components displayed concordant p53 expression patterns in 5 out of 6 cases. However, the aberrant p53 expression pattern, i.e. loss of expression, could not be entirely explained by p53 mutations in the exons examined. Aberrant nuclear ß-catenin expression, as detected in all but one sarcomatoid components, was the result of CTNNB1 mutations only in case No 5 (Table 2). No TERT promoter mutations were detected in four phenotypically diverse samples from two cases harboring TP53 or TP53/CTNNB1 mutations, respectively.
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Discussion
Adrenocortical carcinomas (ACCs) with sarcomatous areas are rare tumors of unknown molecular pathogenesis with respect to associated genetic alterations and potential clonal relationships between the phenotypically diverse tumor components. Herein, we provide novel mutational findings and molecular evidence of monoclonal origin. In addition, we elucidate the immunohistochemical profile as well as mechanisms potentially conferring such a highly aggressive biological behavior to these rare tumor variants, which are enriched for EMT-related markers and stem cell factors.
The shared presence of TP53 and CTNNB1 gene mutations in both the epithelial and sarcomatoid compartments supports a common clonal origin at least in a subset of sarcomatoid ACCs. This is consistent with various molecular genetic studies, i.e. targeted NGS, mutational analysis, cytogenetic analysis, comparative genomic hybridization (CGH) analysis, as well as microdissection-based allelotyping/ loss of heterozygosity (LOH) analysis, in primary cutaneous carcinosarcomas [31-32], hepatic carcinosarcoma [33-34], maxillary carcinosarcoma [35] carcinosarcomas of the female genital tract [36-37], breast carcinosarcomas [38-39] and/or metaplastic breast carcinomas [40-41], salivary gland carcinosarcomas [42-43], sarcomatoid urothelial carcinomas of the urinary bladder [44-46], pharyngeal carcinosarcomas [44], esophageal carcinosarcomas [47], and pulmonary carcinosarcomas [48].
Three tumors harbored TP53 mutations both in epithelial and non-epithelial constituents indicating that these mutations are early driver events in their pathogenesis most likely predisposing tumor cells to acquire additional genetic aberrations that might activate other critical signaling pathways for this particular sarcomatous differentiation lineage. One CTNNB1 mutation concurred with a TP53 mutation in three morphologically diverse tumor components of a single tumor (case No 5). This finding further reinforces a previously proposed association between the status of the TP53 and CTNNB1 genes in adrenocortical tumorigenesis, based on either co-occurences of these mutations or CTNNB1 gains in advanced ACCs [49]. Likewise, aberrant nuclear ß-catenin localization was observed in all TP53-mutated sarcomatous components as well as in all APC-mutated tumor components of case No 3.
By using topographic genotyping with a targeted NGS technique combined with immunohistochemical investigation of EMT-related and stem cell-like markers, we tried to elucidate aspects of the molecular histogenesis of the sarcomatoid ACCs (Supplementary Figure 1). Notably, prior studies in carcinosarcomas, using a targeted NGS approach [33], microsatellite markers [47-48] or through deep sequencing [50] highlighted the complexity of the evolutionary process supporting either a continuous genetic progression model or a branched clonal divergent model with or without subclonal progression.
By immunohistochemistry, we observed a downregulation of Cadherin expression along with an enrichment of various EMT-related markers in sarcomatoid components, implying a potential role for EMT in a subset of sarcomatoid ACCs (Figure 2). Additionally, the sarcomatoid compartments of all cases
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displayed Wnt pathway activation, as evidenced by aberrant nuclear and/or cytoplasmic ß-catenin localization. This could be attributed either to CTNNB1IAPC mutations or potentially to aberrations in other tumor suppressor genes related to the Wnt/B-catenin pathway and/or negative crosstalk between SF-1 and Wnt/B-catenin signaling [51-53]. Mutational inactivation of TP53, accompanied by p53 overexpression, was also a common genetic event (4 out of 6 cases). Given the critical role of the Wnt pathway in EMT [54] and the interplay between a fail-safe program escape and EMT [55-56], these molecular aberrations might mediate an EMT process in a subset of sarcomatoid ACCs. These observations are in agreement with data from mutational and immunohistochemical investigations of metaplastic breast carcinomas [41, 57-58] and primary cutaneous carcinosarcomas [31].
Nestin expression was noted almost exclusively in the sarcomatoid components of all examined cases along with Wnt/B-catenin pathway deregulation. Four tumors displayed SOX2 co-expression in subpopulations of the sarcomatoid components and adjacent epithelial areas (transitional zones in cases No 1 & 5) indicative of an active ongoing process. Of note is that (i) ß-catenin has been shown as an important binding partner of SOX2 and a regulator of its transcriptional activity in a subset of breast cancer cells [59]; (ii) an enhancer region on the nestin gene is dependent on SOX2 binding [60]; and (iii) a biologically significant linkage between nestin and SOX2 expression has been previously reported in human melanomas [61]. Moreover, nestin and/or SOX2 expression is enriched in various tumor types playing a potential role in cancer progession as well as conferring a poor prognosis [61-65]. Although it is unclear whether these participate in EMT [61, 66] or reflect a cancer stem cell phenotype [66], a molecular-driven multifaceted targeted approach (Wnt/ß-catenin signalling pathway, EMT program- associated molecules and nestin) seems to be of great therapeutic interest [64, 67-68].
EMT-associated markers were more significantly expressed in the sarcomatoid areas than in conventional ACCs (lacking sarcomatoid areas) adding further evidence for a role in its pathogenesis. Nestin immunoexpression was detected in 5 out of 38 ACCs (13.5%) and in none of ACAs examined (0%) as compared to other data of 13 out of 16 ACCs (81%) and 2 out of 20 ACAs (10%) [69]. In accordance with EMT-related zinc-finger transcription factor Snail immunoexpression in adrenocortical tumours [70], we detected significant differences in N-/P-Cadherin and ZEB1/Caveolin-1 expression between ACCs and ACAs. Further studies in larger cohorts are warranted to delineate the exact role of EMT in adrenocortical tumorigenesis.
In summary, we have shown that Wnt/B-catenin signaling pathway dysregulation and mutational inactivation of TP53 are common genetic events in adrenal cancers having a sarcomatous component. Targeted NGS approach provided molecular evidence of monoclonal evolution at least in a subset of cases. The term “sarcomatoid ACC” seems more appropriate than carcinosarcoma or others to address such a rare and aggressive variant of ACC. These tumors were enriched in EMT-related markers and stem cell factors, potentially conferring a poor prognosis and being exploited as novel therapeutic targets.
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Figure Legends
Figure 1. H&E staining, ß-catenin and p53 immunoexpression patterns in epithelial (e1 in A-C; e2 in D-F) and sarcomatoid components (s1 in G-I; s2 in J-L) of case No 5. All components harbored an identical CTNNB1 p.S45F mutation displaying nuclear ß-catenin immunoreactivity, whereas only those containing TP53 p.R249W mutations demonstrated strong p53 immunopositivity. Please note that sarcomatoid component (s3) within the inferior vena cava, harboring CTNNB1 p.S45F and TP53 p.R249W mutations and exhibiting similar ß-catenin/p53 immunoexpression patterns to (s2), is not included in the panel.
Figure 2. Immunohistochemical profile of case No 4: Note MMP2 (A)/Caveolin-1 (C)/ Nestin (D) immunoreactivity only in the sarcomatoid component abutting uninvolved pancreatic parenchyma (*) in contrast to N-Cadherin (B) as expressed in the epithelial compartment. ZEB-1 (E) is expressed both in the epithelial and adjacent sarcomatoid areas (transitional zone).
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| Case # | Sex/Age | Location | Symptoms | Size / Weight | Gross Appearance | ACC component | Sarcomatoid component | Clinical Follow-up | Reference |
|---|---|---|---|---|---|---|---|---|---|
| 1 | M/31 | L | Abdominal pain/ no endocrine dysfunction | 12 cm / 620 g | Cystic degenerative cut surface, necrosis, hemorrhage | WS 8 | Spindle cell | Locoregional recurrence with infiltration of the splenic hilum (2 mo) | Sturm [8] |
| 3 months DOD | |||||||||
| 2 | F/75 | L | Abdominal pain/ no endocrine dysfunction | 15 cm | Variegated cut surface with whitish-gray firm solid areas, | WS 7 | Spindle cell | Liver metastases (3 mo) | Coli [9] |
| ACCEPTEDMANUSCRIPT extensive necrosis and cysts | 12 months DOD | ||||||||
| 3 | F/23 | L | Occasional finding, syncronous rectal cancer in pregnancy/ no endocrine dysfunction | 14 cm | Irregular gray surface, central cavity filled with mucinous material | WS 7 ª | Osteosarcoma | widespread metastatic disease b 14 months DOD | Bertolini [11] |
| 4 | M/55 | L | Abdominal pain/ no endocrine dysfunction | 16 cm | Whitish gray cut surface, necrosis, hemorrhage | WS 9c | Spindle cell d | widespread abdominal metastatic disease 4 months DOD | This study |
| 5 | F/70 | R | Abdominal | 15 cm | Cystic cut surface; cysts | WS 8 | Osteosarcoma | Liver | This |
| pain, diarrhea/ | metastasis (2 | ||||||||
| no endocrine dysfunction | filled with clotted blood and viable appearing gray-white to partly yellow- white parenchyma Firm calcific areas Extensive necrosis and hemorhage | Spindle cell d, e | mo) Lung and bone metastases (5 mo) 8 months f DOD | study | |||||
| 6 | M/52 | R | Abdominal | 24 cm/ | Solid and | WS 5 | Spindle cell d | Liver metastasis (two months prior to surgical | This |
| pain, fatigue, malaise, | 3020 g | partially cystic cut surface. Multiple necrotic | study |
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| weight loss/ | areas | resection) |
|---|---|---|
| no endocrine | Pleural | |
| dysfunction | metastasis (4 | |
| mo) |
4.5 months f DOD
Abbreviations: ACC, adrenocortical carcinoma; F, female; M, male; NE; not evaluated; L, left; R, right; WS, Weiss Score
ª a conventional ACC with adipocytic-like areas displaying MDM2 gene polysomy as detected by FISH (experimental details available upon request) co-existing with a metastatic rectal adenocarcinoma
b given the synchronous occurrence of a rectal adenocarcinoma, it becomes difficult to define the exact origin of the metastatic foci; due to the histopathologically proven metastasis of the rectal cancer to the adrenal gland, the patient was treated as having metastatic rectal cancer and thus mitotane was not an option
” this case was originally reported as pheochromocytoma, due to a pheochromocytoma-like component comprising medium-sized cells, displaying scant nuclear atypia and a weakly eosinophilic cytoplasm, arranged in a nested or trabecular pattern. The diagnosis was amended (sarcomatoid ACC) following consultation.
d the sarcomatoid component is estimated approximately >10% of the total tumour extent
e two phenotypically diverse spindle cell components were identified (Figure 1)
postoperative time of death; Cases 5/6 were both treated with surgical resection and mitotane
ACCEPTEREN en todo el cole, powerful, HE HE . .
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Table 2. Summary of pathogenic variants as detected by targeted NGS approach & B-catenin/P53 IHC as evaluated in 16 morphologically distinct tumour components from 6 sarcomatoid ACCs
| Case # | Pathogenic Mutations | ß-catenin IHC | P53 IHC MANUSCRIPT |
|---|---|---|---|
| 1 (e) | --- | m | nor |
| 1 (s) | TP53 | n & c | + |
| c.973G>T p.G325X | |||
| (exon 9) | |||
| 2 (e) | --- | m | |
| 2 (s) | --- | C | |
| 3 (e1) | a | n | |
| 3 (e2) | a | n | |
| 3 (s) | --- a | n | |
| 4 (e) | TP53 | m | + |
| c.995T>G p.I332S | |||
| (exon 10) | |||
| 4 (s) | TP53 | n | + |
| c.995T>G p.1332S | |||
| (exon 10) | |||
| 5 (e1) | CTNNB1 | n | + |
| DA CONTROLLAREPTED c.134C>T p.S45F | |||
| (exon 3) | |||
| TP53 | |||
| c.745A>T p.R249W | |||
| (exon 7) | |||
| 5 (e2) | CTNNB1 | n & m | - |
| c.134C>T p.S45F | |||
| (exon 3) | |||
| 5 (s1) | CTNNB1 | n | - |
| c.134C>T p.S45F |
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(exon 3)
5 (s2)
CTNNB1
n & c
+
c.134C>T p.S45F
+ Pasta RECEPTED MANUSCRIPT
(exon 3)
TP53
c.745A>T p.R249W
(exon 7)
5 (s3)
CTNNB1
n & c
c.134C>T p.S45F
(exon 3)
TP53
c.745A>T p.R249W
(exon 7)
6 (e)
TP53
m +
c.743G>A p.R248Q
(exon 7)
6 (s)
TP53
n
+
c. 743G>A p.R248Q
(exon 7)
Abbreviations: +, overexpression; - , loss of expression; c, cytoplasmic; e, epithelial component; IHC, immunohistochemistry; m, membranous; nor; normal; n, nuclear; s, sarcomatoid component
a known germline APC mutation [11]
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| Antibodies | Case # 1 | Case # 2 | Case # 3 | Case # 4 ACCEPTED MANUSCRIPT | Case # 5 | Case # 6 |
|---|---|---|---|---|---|---|
| [8] | [9] | [11] | ||||
| e S | e S | e2 S | e S | e2 s2 | e s | |
| E- Cadherin | - | + | - | - - | ||
| P- | a - | + | - | - - | ||
| Cadherin | ||||||
| N- | - | + | + | - | - | |
| Cadherin | ||||||
| MMP-2 | - | + | + | + | + - | |
| MMP-9 | + - | + | + | + - | ||
| ZEB-1 | + - | + | + | + + | ||
| ZEB-2 | - | - | - - | |||
| SLUG | + | + + | + - | |||
| Caveolin- | + - | + | + - | |||
| 1 | ||||||
| OCT3/4 | - | - | - - | |||
| NANOG | - | - | - - | |||
| CD133 | - - | - - | - - | - - | - - | - - |
| LIN28 | + + | - - | a a - - | - - | + - | a + - |
| SOX2 | c + - | - - | - | - - | c + - | + - |
| SOX17 | - - | - - | - - | - - | - - | + - |
| Nestin | c + - | + - | + + | + - | + - | + - |
Abbreviations: EMT, epithelial-mesenchymal transition; e, epithelial component; s, sarcomatoid component
ª only cytoplasmic localization
b epithelial areas displayed also N-Cadherin immunonegativity
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’ other epithelial areas adjacent to sarcomatous areas (transitional areas) displayed immunoreactivity
d osteoblasts displayed immunoreactivity, while osteocytes immunonegativity
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A
B
C
D
E
F
5
G
H
I
J
K
L
ACC
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C
A
*
D
B
E
ACCER