- Email: [email protected]
Diagnostic utility of cyclin D1 in the diagnosis of small round blue cell tumors in children and adolescents
Diagnostic Utility of Cyclin D1 in the Diagnosis of Small Round Blue Cell Tumors in Children and Adolescents Gaetano Magro MD, PhD, Lucia Salvatorelli MD, Rita Alaggio MD, Velia D’Agata, Ferdinando Nicoletti, Andrea Di Cataldo, Rosalba Parenti PII: DOI: Reference:
S0046-8177(16)30264-7 doi: 10.1016/j.humpath.2016.07.038 YHUPA 4038
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
22 June 2016 20 July 2016 22 July 2016
Please cite this article as: Magro Gaetano, Salvatorelli Lucia, Alaggio Rita, D’Agata Velia, Nicoletti Ferdinando, Di Cataldo Andrea, Parenti Rosalba, Diagnostic Utility of Cyclin D1 in the Diagnosis of Small Round Blue Cell Tumors in Children and Adolescents, Human Pathology (2016), doi: 10.1016/j.humpath.2016.07.038
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Diagnostic Utility of Cyclin D1 in the Diagnosis of Small Round Blue Cell Tumors in Children and Adolescents
T
Gaetano Magro MD, PhDa,*, Lucia Salvatorelli MDa, Rita Alaggio MDb, Velia D’Agatac,
RI P
Ferdinando Nicolettid, Andrea Di Cataldoe, Rosalba Parentif
a
SC
[AU: degrees?]
Department of Medical and Surgical Sciences and Advanced Technologies, G.F. Ingrassia,
MA NU
Azienda Ospedaliero-Universitaria “Policlinico-Vittorio Emanuele,” Anatomic Pathology Section, School of Medicine, University of Catania, Catania, Italy b
Department of Medicine-DIMED, Pathology Unit, University of Padova, Italy
c
Department of Biomedical and Biotechnological Sciences, Section of Human Anatomy and
ED
Histology, University of Catania, Catania, Italy. Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy
e
Pediatric Hematology and Oncology Unit, Department of Pediatrics, University of Catania
PT
d
Physiology Section, Department of Biomedical and Biotechnological Sciences, University of
AC
f
CE
Catania, Italy
Catania, Catania, Italy [AU: postal codes?]
*Address for correspondence: Prof. Gaetano Magro MD, PhD Department of Medical and Surgical Sciences and Advanced Technologies, G.F. Ingrassia, Azienda Ospedaliero-Universitaria “Policlinico-Vittorio Emanuele,” Anatomic Pathology Section, School of Medicine, University of Catania, Via S. Sofia 87, 95123 Catania, Italy E-mail address: [email protected]
ACCEPTED MANUSCRIPT Short title: Cyclin D1 in small round blue cell tumors Disclosures: The authors have no conflicts of interest or funding disclosures.
T
Keywords: Cyclin D1; Immunohistochemistry; Small round blue cell tumors; Children;
RI P
Adolescents
SC
Abstract Small round blue cell tumors (SRBCTs) of children and adolescents are often diagnostically challenging lesions. With the increasing diagnostic approach based on small biopsies,
MA NU
there is the need of specific immunomarkers that can help in the differential diagnosis among the different tumor histotypes to assure the patient a correct diagnosis for proper treatment. Based on our recent studies showing cyclin D1 overexpression in both Ewing’s sarcoma/primitive peripheral neuroectodermal tumor (EWS/pPNET) and peripheral neuroblastic tumors (neuroblastoma and
ED
ganglioneuroblastoma), we immunohistochemically assessed cyclin D1 immunoreactivity in 128
PT
cases of SRBCTs in children and adolescents in order to establish its potential utility in the differential diagnosis. All cases of EWS/pPNET and the undifferentiated/poorly differentiated
CE
neuroblastomatous component of all peripheral neuroblastic tumors exhibited strong and diffuse
AC
nuclear staining (>50% of neoplastic cells) for cyclin D1. In contrast, this marker was absent from rhabdomyosarcoma (regardless of subtype) and lymphoblastic lymphoma (either B- or T-cell precursor cells), while it was only focally detected (<5% of neoplastic cells) in some cases of Wilms tumor (blastemal component) and desmoplastic small round cell tumor. Our findings suggest that cyclin D1 can be exploitable as a diagnostic adjunct to conventional markers in confirming the diagnosis of EWS/pPNET or neuroblastoma/ganglioneuroblastoma. Its use in routine practice also may be helpful for those cases of SRBCT with undifferentiated morphology that are difficult to diagnose following application of the conventional markers.
ACCEPTED MANUSCRIPT 1. Introduction Small round blue cell tumors (SRBCTs) of children and adolescents are diagnostically
T
challenging lesions. Currently, their diagnosis is mostly based on morphology,
RI P
immunohistochemical analyses, as well as additional ancillary studies (cytogenetic and molecular studies). Diagnostic difficulties arise because some tumors show overlapping morphology and at
SC
least partly immunohistochemical features, or may present unusual clinical and/or pathological features [1-6]. As diagnosis based on small biopsies is increasing in daily practice, SRBCTs do
MA NU
represent a potential pitfall, especially if pathologists are not familiar with these entities [2,4,7-9]. Given the differences in disease biology, clinical behavior and therapeutic approach among the different histotypes of SRBCTs, an accurate diagnosis is mandatory for assuring the patient correct prognostic information and proper treatment. Accordingly, there is the need to identify novel
ED
specific immunomarkers that could be used successfully as cheap, fast and easily accessible
PT
confirmatory diagnostic tests in daily practice [2]. Cyclin D1 is a G1-specific cyclin that plays a crucial role in regulating cell cycle
CE
progression in G1/S transition, and therefore it is not at all surprising if it is overexpressed in
AC
several malignant tumors, including carcinomas, sarcomas and lymphomas [1,10]. Depending on tumor histotype, the altered expression of cyclin D1 is mainly due to different genomic alterations, including chromosomal translocation or amplification, post-transcriptional regulation or posttranslational protein stabilization [11-13]. Immunohistochemical expression of cyclin D1 has not been performed systematically in SRBCTs of children and adolescents. In this regard, an immunohistochemical study reported cyclin D1 expression only in 42% of cases of Ewing’s sarcoma/primitive peripheral neuroectodermal tumor (EWS/pPNET) [14], while another study, performed on peripheral neuroblastic tumors, found cyclin D1 overexpression in neuroblasts, in contrast to low expression in all cell types of ganglioneuromas [15]. Recently, we have found that cyclin D1 is overexpressed by soft tissue EWS/pPNET in children and adolescents, while it is absent from rhabdomyosarcoma, despite the subtype (embryonal, spindle cell/sclerosing, alveolar
ACCEPTED MANUSCRIPT subtypes) [16]. In another immunohistochemical study dealing with peripheral neuroblastic tumors of childhood, we showed that cyclin D1 expression was limited to the neuroblastic cell component
T
of neuroblastomas and ganglioneuroblastomas, whereas it is lacking, or only focally detectable, in
RI P
the maturing/mature ganglion cell component of differentiating neuroblastomas, ganglioneuroblastomas, and ganglioneuromas [17]. Notably, we also emphasize that cyclin D1 in
SC
neuroblastic tumors recapitulates its developmental expression [17]. These immunohistochemical results have been supported by publicly available oligonucleotide microarrays showing significant
MA NU
upregulation of cyclin D1 in both Ewing’s sarcoma and neuroblastoma as compared to normal tissues or other malignant tumors [18,19].
These promising results prompted us to evaluate the potential diagnostic utility of cyclin D1 as an immunomarker in the differential diagnosis of the most common tumors that usually exhibit
ED
small round cell morphology in children and adolescents. A large series of 128 cases of SRBCTs,
PT
including EWS/pPNET, rhabdomyosarcoma, Wilms tumor, lymphoblastic lymphoma and
CE
desmoplastic small round cell tumor, were immunohistochemically tested for cyclin D1 expression.
AC
2. Materials and methods 2.1. Ethics statement
The present research complied with the Helsinki Declaration and was approved by the Human Ethics Committee and the Research Ethics Committee of the University of Catania. All patients provided written informed consent according to institutional guidelines. Patients were informed that the resected specimens were stored by the sections of Anatomic Pathology of both Universities of Catania and Padova and might potentially be used for scientific research, and that their privacy would be maintained.
ACCEPTED MANUSCRIPT 2.2. Tissue specimens The cases were retrospectively retrieved from the surgical pathology archives of the section
T
of Anatomic Pathology, G.F. Ingrassia Department of Medical, Surgical, and Advanced
RI P
Technologies, at University of Catania and from the surgical pathology archives of the Italian reference center for pediatric soft tissue sarcomas at the University of Padova. Clinical data were
SC
obtained from the original pathology reports. Hematoxylin and eosin–stained slides (H&E) and a variable number of slides stained with several antibodies were available for each case. All the H&E
MA NU
slides were reviewed by two expert surgical pathologists (R.A. and G.M.) of the GIPOP (Italian Group of Oncologic Pediatric Pathology), and the diagnoses were histologically confirmed using the current well-established morphological criteria and immunohistochemical features [20-24]. Molecular data (fusion gene products) by reverse-transcription polymerase chain reaction (RT–
ED
PCR) were also available for some cases. At least one representative formalin-fixed, paraffin-
PT
embedded block was available for immunohistochemical analyses. We selected 30 cases of soft tissue EWS/pPNET, 26 cases of rhabdomyosarcoma, 41 cases
CE
of peripheral neuroblastic tumors, 15 cases of Wilms tumor (WT), 6 cases of desmoplastic small
AC
round cell tumor (DSRCT) and 10 cases of lymphoblastic lymphomas (pre-B or pre-T precursor cells). All tissue samples were derived from primary tumors of untreated patients. Most of the cases of EWS/pPNET, rhabdomyosarcoma and peripheral neuroblastic tumors were included in previously published studies [16,17]. Patients with EWS/pPNET diagnosis were 21 males and 9 females, with ages ranging from 4 to 19 years (median age 11.5 years). Tumors were from soft tissues of extremities (14 cases), chest wall (7 cases), paravertebral (5 cases), intra-abdominal (2 cases) and pelvic (1 case) regions, and retroperitoneum (1 case). Most tumors (26/30) were derived from incisional biopsies, with the exception of 4 cases (2 cases from surgically excised specimen; 2 cases from needle core biopsy). As CIC-DUX4 and BCOR fusion sarcomas with Ewing-like morphology are emerging entities [2529], we included in this study exclusively classic-type EWS/pPNET. Histologically, tumors were
ACCEPTED MANUSCRIPT composed of uniform small round blue cells arranged in a vaguely lobular to sheet-like growth pattern, and they were strongly and diffusely stained (along the cell membrane of neoplastic cells)
T
with CD99. None of the cases selected showed atypical morphology (heterogeneity in tumor cell
RI P
size and shape, nuclear enlargement, irregular nuclear contours, frequent prominent nucleoli, diffuse myxoid change, delicate arborizing capillary network) and/or ambiguous CD99
SC
immunostaining (focal, heterogeneous, or incomplete staining along the cell membrane). All cases were negative for desmin, myogenin, MyoD1, NB84, TdT, CD56, α-smooth muscle actin, pan-
MA NU
cytokeratins, S100 protein, and WT1. FLI-1 was positive in the cases that were originally tested for this antibody (15/15 cases). Molecular data were also available for 18 cases of EWS/pPNET. RT– PCR identified the EWS-FL1 fusion product.
The cases of rhabdomyosarcoma were represented by 10 cases of embryonal
ED
rhabdomyosarcoma, 2 cases of spindle cell/sclerosing rhabdomyosarcoma and 14 cases of alveolar
PT
rhabdomyosarcoma. Most tumors (18/26) derived from incisional biopsies, while the remaining cases (8/26) were from surgically excised specimens. The patients affected with embryonal
CE
rhabdomyosarcoma were 7 males and 3 females, with ages ranging from 1 to 11 years (median age
AC
6.5 years). Tumors occurred in the orbit (2 cases), palate (2 cases), bladder (2 cases), trunk (1 case), paratesticular (1 case), nasopharynx (1 case), auditory canal (1 case). The patients affected with spindle cell/sclerosing rhabdomyosarcoma were 2 males (9 and 11 years), and both tumors occurred in the palate. The patients affected with alveolar rhabdomyosarcoma were 7 males and 7 females, with ages ranging from 1 to 12 years (median age 9.7 years). Tumors occurred in soft tissues of abdominal wall (4 cases), extremities (3 cases), testis (3 cases), pelvic region (2 case), orbit (1 case) and groin (1 case). All cases of rhabdomyosarcomas, regardless of histological subtype, were positive (albeit with variable extension) for desmin, myogenin and MyoD1, while they were negative for CD99, NB84, CD56, FLI-1, TdT, WT1 (negative nuclear staining), α-smooth muscle actin, S-100 protein, and pan-cytokeratins. Molecular data were also available in 9 cases of alveolar
ACCEPTED MANUSCRIPT rhabdomyosarcoma. RT-PCR identified the PAX3/FKHR fusion product in 6 cases, whereas the other 3 cases tested were fusion negative.
T
The cases of peripheral neuroblastic tumors were classified based on well-established
RI P
morphological criteria [22,23]: (i) 22 Schwannian stroma-poor tumors (1 undifferentiated neuroblastoma, 19 poorly differentiated neuroblastomas, 2 differentiating neuroblastomas); (ii) 14
SC
Schwannian stroma-rich tumors (12 intermixed ganglioneuroblastomas, 2 nodular ganglioneuroblastomas); (iii) 5 Schwannian stroma-predominant tumors (5 mature
MA NU
ganglioneuromas). The patients were 23 males and 18 females, with ages ranging from 1 month to 9 years (median age 4.2 years). Tumors occurred in adrenal gland, retroperitoneum, superior and posterior mediastinum.
Patients with WT diagnosis were 12 females and 3 males, with ages ranging from 1 to 8
ED
years (median age 4.6 years). All cases but one derived from nephrectomy specimens.
PT
Histologically, 10 cases showed a triphasic pattern, while 4 cases were blastemal predominant (only focal epithelial and stromal components were identified). The single case of WT, in which only
CE
biopsy was available, showed exclusively a blastematous component. In all cases examined,
AC
anaplasia—namely, large pleomorphic cells with marked nuclear atypia and atypical (multipolar) mitotic figures—was not observed. Patients with DSRCT diagnosis were 4 males and 2 females, with ages ranging from 8 to 18 years. All tumors were from intra-abdominal locations and immunohistochemically coexpressed cytokeratins, vimentin, desmin, and WT1 (carboxy-terminus WT1 antibody). They were negative for myogenin, MyoD1, CD99, TdT, and NB84. Molecular data were also available in 4 cases. RT– PCR identified the EWSR-WT1 fusion transcript. Patients with lymphoblastic lymphomas (7 patients with B-precursor cell lymphomas; 3 patients with T-precursor cell lymphomas) were 8 males and 2 females, with ages ranging from 1 to 19 years (median age 6 years). Precursor T-cell lymphomas were located in the anterior mediastinum, while the precursor-B-cell lymphomas presented as enlarged cervical (2 cases) and
ACCEPTED MANUSCRIPT axillary (1 case) lymph nodes, testicular masses (2 cases), intra-abdominal mass (1 case) and palate mass (1 case).
T
Immunohistochemical analyses were performed as previously reported in detail [16,17].
RI P
Briefly, after appropriate deparaffinization and pretreatments, sections were incubated with anti– cyclin D1 (SP4, NeoMarkers [AU: supplier location?]; prediluted antibody) at pH 6.0 for 60 min at
SC
room temperature. Microwave pretreatment was crucial to enhance the staining in all examined samples. Accordingly, all sections were pretreated with citrate buffer (pH 6.0) and exposed to
MA NU
radiation in a microwave oven. To reduce the commonly seen nonspecific immunoreactivity due to endogenous biotin, sections were pretreated with 10 mg/mL of ovalbumin in phosphate-buffered saline (PBS) followed by 0.2% biotin in PBS, each for 15 min at room temperature. Bound antibody was revealed by incubation with 3,3ʹ-diaminobenzidine (Sigma–Aldrich, St. Louis MO, USA) in
ED
0.01% H2O2 for 5 min at room temperature. Sections were then counterstained with hematoxylin,
PT
dehydrated, and mounted. Negative controls, involving the omission of the primary antibody, were included. The percentage of positively stained cells was assessed by semiquantitative optical
CE
analysis according to a four-tiered system (<1% positive cells, negative staining; 1%–10% positive
AC
cells, focal staining; 11%–50% positive cells, heterogeneous staining; >50% positive cells, diffuse staining). Staining intensity was graded as weak, moderate, or strong intensity.
3. Results Immunohistochemical results are summarized in the Table. Among SRBCTs, only EWS/pPNET and neuroblastic tumors showed diffuse expression of cyclin D1. All cases (30/30) of EWS/pPNET exhibited a strong and diffuse nuclear immunoreactivity for cyclin D1 (>50% of stained neoplastic cells) regardless of the tissue specimen tested (incisional, core needle biopsy, surgical excision) (Fig. 1B and D). The percentage of positive cells varied in the different cases, ranging from 60% to 95%. As expected, cyclin D1 was exclusively confined to the nuclei of tumor cells, with no immunoreactivity in their cytoplasm (Fig. 1B, D, E).
ACCEPTED MANUSCRIPT As far as neuroblastic tumors are concerned, the expression of cyclin D1 varied among the different cell types. We observed a diffuse (>70% of neoplastic cells) cyclin D1 nuclear staining
T
restricted to undifferentiated or poorly differentiated neuroblastic cells, while it was absent or only
RI P
focally (<5% of neoplastic cells) found in the neuroblastic cells with clear-cut ganglion cell differentiation, as well as in the mature ganglion cells (Figs. 2 and 3). Similar results were obtained
SC
regardless of the tissue specimen tested (incisional, core needle biopsy, surgical excision). Briefly, the highest cyclin D1 expression was found in both undifferentiated/poorly differentiated
MA NU
neuroblastomas (Fig. 2C and F, Fig. 3C) and in the neuroblastic component of ganglioneuroblastomas (both intermixed and nodular types) (Fig. 3E and F). No cyclin D1 expression was detected in the Schwann cell component of both ganglioneuroblastomas and ganglioneuromas.
ED
The other SRBCTs were negative or only focally positive for cyclin D1. Notably, no case of
PT
rhabdomyosarcoma (0/26), regardless histologic subtypes (Fig. 4B and D), or lymphoblastic lymphoma (0/10) (Fig. 4F) showed cyclin D1 immunoreactivity. Blastemal areas of WTs were
CE
negative (9/15 cases) or only focally (5%-10% stained cells) positive (6/15 cases) for cyclin D1
AC
(Fig. 4H). Similarly, 3 cases of DSRCT were negative (Fig. 4L), while the remaining 3 cases exhibited focal immunoreactivity (5%-10% stained cells). In all cases of SRBCTs tested, cyclin D1 variably stained the nuclei of endothelial cells of intratumoral blood vessels and served as internal control (Fig. 4D).
4. Discussion The differential diagnosis of SRBCTs can be challenging, especially in some clinicopathologic contexts. This is particularly true when SRBCTs are located in the abdominal cavity, because they can present overlapping clinicopathologic features. In daily practice, it is not rare that radiological imaging is not conclusive in identifying the organ (adrenal vs kidney; kidney vs retroperitoneum) where a tumor mass arises in children or adolescents. In this regard, a WT may
ACCEPTED MANUSCRIPT be so large in size that it could be difficult to establish with certainty its origin from the kidney. In addition, there is the possibility that other SRBCTs, such as EWS/pPNET or DSRCT, may
T
primarily arise in visceral organs, including kidney and retroperitoneum [3,24,30]. Therefore,
RI P
distinguishing a blastemal-predominant WT from other SRBCTs may be extremely difficult, especially if the pathologist is evaluating small biopsies from a renal/intra-abdominal mass. In
SC
addition, the presence of epithelial elements in the context of an undifferentiated cellular component, although highly suggestive, is not diagnostic of WT, as some cases of DSRCT may
MA NU
exhibit glandular differentiation. Similarly diagnostic problems may arise if bioptic material is obtained from an undifferentiated or poorly differentiated neuroblastoma located in the abdominal cavity. In fact, the presence of small round blue cells as the only cytotype, together with the absence of neuropil, makes very difficult the morphological distinction of undifferentiated neuroblastoma
ED
from other SRBCTs, especially EWS/pPNET and lymphoblastic lymphoma. Based on these
PT
considerations, the availability of highly sensitive and specific immunomarkers for the different tumor histotypes is crucial for an accurate diagnosis of the SRBCTs in children and adolescents.
CE
Our preliminary results showed that cyclin D1 is overexpressed in both soft tissue
AC
EWS/pPNET and neuroblastomas/ganglioneuroblastomas of children and adolescents [16,17]. The goal of the present study was to validate these results by assessing the potential diagnostic utility of cyclin D1 in the differential diagnosis of the most common SRBCTs. We showed that all cases (30/30) of soft tissue EWS/pPNET and the undifferentiated/poorly differentiated neuroblastomatous component of peripheral neuroblastic tumors exhibited a strong and diffuse (>50% of stained neoplastic cells) nuclear staining for cyclin D1. In contrast, this marker was absent from rhabdomyosarcoma (regardless of the subtype) and lymphoblastic lymphoma (either B- or T-cell precursor cells). Cyclin D1 immunoreactivity was also absent, or only focally detected (<5% of neoplastic cells), in the blastemal component of WT and in the neoplastic cells of DSRCT. Two recent studies on pediatric renal tumors obtained similar results, with absent to focal expression of
ACCEPTED MANUSCRIPT cyclin D1 in a small series of WT [31,32]. Our results suggest that cyclin D1 is a useful marker in separating EWS/pPNET and neuroblastomas/ganglioneuroblastomas from other SRBCTs.
T
As the immunohistochemical expression of cyclin D1 has been documented in a variety of
RI P
malignant tumors [33-38], its use as a single diagnostic immunomarker should be avoided. However, we suggest that a diffuse nuclear expression of cyclin D1, in the appropriate
SC
clinicopathologic context, can be exploitable as a diagnostic adjunct to conventional markers, such as CD99 and FLI-1 or NB84 and CD56, in confirming the diagnosis of EWS/pPNET or
MA NU
neuroblastoma/ganglioneuroblastoma, respectively. In addition cyclin D1 immunoreactivity may have an informative value when dealing with a SRBCT exhibiting undifferentiated morphology, which is difficult to diagnose even following application of the conventional markers (ambiguous staining). Diffuse and strong cyclin D1 expression should prompt pathologists to perform genetic
ED
and/or molecular studies as confirmatory tests for EWS/pPNET or
PT
neuroblastomas/ganglioneuroblastomas.
Although the main purpose of this study was to evaluate the use of cyclin D1 in the
CE
differential diagnosis of SRBCTs in children and adolescents, its diffuse expression either in
AC
EWS/pPNETs or neuroblastomas/ganglioneuroblastoma provides the rationale for exploiting cyclin D1 as a target for new strategic therapies. In this regard, there are promising phase I and II clinical trials based on cyclin D1 overexpression [11]. The mechanisms proposed range from cyclin D1 downregulation to inhibition of cyclin D1 action. The most immediately feasible approach is directed to inhibition of the cyclin D1–CDK4/CDK6 complex to induce a decrease of cell proliferation. In several emerging studies, cyclin D1 is the primary target of compounds such as cytotoxics, radiotherapy, and mTOR inhibitors able to determine the degradation of overexpressing cyclin D1 cancer cells [12]. Another proposed mechanism is the use of competitive inhibitors for specific domains in cyclin D1 or by substrate mimetics [12]. To date, the use of only one of these methods has had limited success; thus the trend would be the use of combined therapies, for
ACCEPTED MANUSCRIPT example, the most promising combination is among the inhibitors of CDKs and cytotoxic drugs
T
[12].
RI P
Acknowledgments
The authors thank Dr.ssa Spoto Graziana for her excellent technical assistance. The paper was
SC
supported by the Italian funding FIR2014.
MA NU
References
[1] Coffin CM, Alaggio R, Dehner LP. Some general considerations about the clinicopathologic aspects of soft tissue tumors in children and adolescents. Pediatr Dev Pathol 2012;15:11-25. [2] Magro G, Longo FR, Angelico G, Spadola S, Amore FF, Salvatorelli L. Immunohistochemistry
ED
as potential diagnostic pitfall in the most common solid tumors of children and adolescents.
PT
Acta Histochem 2015;117:397-414.
[3] Arnold MA, Schoenfield L, Limketkai BN, Arnold CA. Diagnostic pitfalls of differentiating
CE
desmoplastic small round cell tumor (DSRCT) from Wilms tumor (WT): overlapping
AC
morphologic and immunohistochemical features. Am J Surg Pathol 2014;38:1220-6. [4] Alaggio R, Boldrini R, Di Venosa B, Rosolen A, Bisogno G, Magro G. Pediatric extra-renal rhabdoid tumors with unusual morphology: a diagnostic pitfall for small biopsies. Pathol Res Pract 2009;205:451-7. [5] Magro G, Greco P, Alaggio R, Gangemi P, Ninfo V. Polypoid angiomyofibroblastoma-like tumor of the oral cavity: a hitherto unreported soft tissue tumor mimicking embryonal rhabdomyosarcoma. Pathol Res Pract 2008;204:837-43. [6] Bahrami A, Gown AM, Baird GS, Hicks MJ, Folpe AL. Aberrant expression of epithelial and neuroendocrine markers in alveolar rhabdomyosarcoma: a potentially serious diagnostic pitfall. Mod Pathol 2008;21:795–806.
ACCEPTED MANUSCRIPT [7] Wang H, Li F, Liu J, Zhang S. Ultrasound-guided core needle biopsy in diagnosis of abdominal and pelvic neoplasm in pediatric patients. Pediatr Surg Int 2014;30:31-7.
T
[8] Gustafson S, Medeiros LJ, Kalhor N, Bueso-Ramos CE. Anaplastic large cell lymphoma:
RI P
another entity in the differential diagnosis of small round blue cell tumors. Ann Diagn Pathol 2009;13:413-27.
SC
[9] Fowler DJ, Malone M, Chisholm J, Roebuck D, Sebire NJ. Primary thoracic myxoid variant of extrarenal rhabdoid tumor in childhood. Fetal Pediatr Pathol 2006;25:159-68.
MA NU
[10] Zukerberg LR, Yang WI, Arnold A, Harris NL. Cyclin D1 expression in non-Hodgkin's lymphomas. Detection by immunohistochemistry. Am J Clin Pathol 1995;103:756-60. [11] Musgrove EA, Caldon CE, Barraclough J, Stone A, Sutherland RL. Cyclin D as a therapeutic target in cancer. Nat Rev Cancer 2011;11:558-72.
ED
[12] Kim JK, Diehl JA. Nuclear cyclin D1: an oncogenic driver in human cancer. J Cell Physiol
PT
2009; 220:292-6.
[13] Pestell RG. New roles of cyclin D1. Am J Pathol 2013;183:3-9.
CE
[14] Fuchs B, Inwards CY, Janknecht R. Vascular endothelial growth factor expression is up-
AC
regulated by EWS-ETS oncoproteins and Sp1 and may represent an independent predictor of survival in Ewing’s sarcoma. Clin Cancer Res 2004;10:1344-53. [15] Molenaar JJ, Ebus ME, Koster J, et al. Cyclin D1 and CDK4 activity contribute to the undifferentiated phenotype in neuroblastoma. Cancer Res 2008;68:2599-609. [16] Magro G, Brancato F, Musumeci G, Alaggio R, Parenti R, Salvatorelli L. Cyclin D1 is a useful marker for soft tissue Ewing’s sarcoma/peripheral primitive neuroectodermal tumor in children and adolescents: a comparative immunohistochemical study with rhabdomyosarcoma. Acta Histochem 2015;117:460-7. [17] Magro G, Salvatorelli L, Di Cataldo A, Musumeci G, Spoto G, Parenti R. Cyclin D1 in human neuroblastic tumors recapitulates its developmental expression: An immunohistochemical study. Acta Histochem 2015;117:415-24.
ACCEPTED MANUSCRIPT [18] Fagone P, Nicoletti F, Vecchio GM, Parenti R, Magro G. Cyclin D1 in pediatric neuroblastic tumors: a microarray analysis. Acta Histochem 2015;117:820-3.
T
[19] Fagone P, Nicoletti F, Salvatorelli L, Musumeci G, Magro G. Cyclin D1 and Ewing’s
RI P
sarcoma/PNET: A microarray analysis. Acta Histochem 2015;117:824-8. [20] Parham DM, Alaggio R, Coffin CM. Myogenic tumors in children and adolescents. Pediatr
SC
Dev Pathol 2012;15:211-38.
[21] Tsokos M, Alaggio RD, Dehner LP, Dickman PS. Ewing sarcoma/peripheral primitive
MA NU
neuroectodermal tumor and related tumors. Pediatr Dev Pathol 2012;15:108-26 [22] Joshy VV, Silverman JF. Pathology of neuroblastic tumors. Semin Diagn Pathol 1994;11:107– 11.
[23] Shimada H, Ambros IM, Dehner LP, et al. The International Neuroblastoma Pathology
ED
Classification (the Shimada system). Cancer 1999;86:364-72.
PT
[24] da Silva RC, Medeiros Filho P, Chioato L, Silva TR, Ribeiro SM, Bacchi CE. Desmoplastic small round cell tumor of the kidney mimicking Wilms tumor: a case report and review of the
CE
literature. Appl Immunohistochem Mol Morphol 2009;17:557-62.
AC
[25] Graham C, Chilton-MacNeill S, Zielenska M, Somers GR. The CIC-DUX4 fusion transcript is present in a subgroup of pediatric primitive round cell sarcomas. HUM PATHOL 2012;43:180-9. [26] Italiano A, Sung YS, Zhang L, et al. High prevalence of CIC fusion with double-homeobox (DUX4) transcription factors in EWSR1-negative undifferentiated small blue round cell sarcomas. Genes Chromosomes Cancer 2012;51:207-18. [27] Antonescu C. Round cell sarcomas beyond Ewing: emerging entities. Histopathology 2014;64:26-37. [28] Machado I, Navarro S, Llombart-Bosch A. Ewing sarcoma and the new emerging Ewing-like sarcomas: (CIC and BCOR-rearranged-sarcomas). A systematic review. Histol Histopathol. 2016;31:1169-91.
ACCEPTED MANUSCRIPT [29] Kao YC, Sung YS, Zhang L, et al. Recurrent BCOR internal tandem duplication and YWHAENUTM2B fusions in soft tissue undifferentiated round cell sarcoma of infancy: overlapping
T
genetic features with clear cell sarcoma of kidney. Am J Surg Pathol 2016;40:1009-20.
RI P
[30] Wang LL, Perlman EJ, Vujanic GM, et al. Desmoplastic small round cell tumor of the kidney in childhood. Am J Surg Pathol 2007;31:576-84.
SC
[31] Jet Aw S, Hong Kuick C, Hwee Yong M, et al. Novel karyotypes and cyclin D1 immunoreactivity in clear cell sarcoma of the kidney. Pediatr Dev Pathol 2015;18:297-304.
MA NU
[32] Mirkovic J, Calicchio M, Fletcher CD, Perez-Atayde AR. Diffuse and strong cyclin D1 immunoreactivity in clear cell sarcoma of the kidney. Histopathology 2015;67:306-12. [33] Diehl JA. Cycling to cancer with cyclin D1. Cancer Biol Ther 2002;1:226–31. [34] Fu M, Wang C, Li Z, Sakamaki T, Pestell RG. Minireview cyclin D1: normal and abnormal
ED
functions. Endocrinology 2004;145:5439–47.
PT
[35] Sutherland RL, Musgrove EA. Cyclins and breast cancer. J Mammary Gland Biol Neoplasia 2004;9:95–104.
CE
[36] Knudsen KE, Diehl JA, Haiman CA, Knudsen ES. Cyclin D1: polymorphism, aberrant splicing
AC
and cancer risk. Oncogene 2006;25:1620–8. [37] Musgrove EA. Cyclins: roles in mitogenic signaling and oncogenic transformation. Growth Factors 2006;24:13–9. [38] Alao JP. The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention. Mol Cancer 2007;2:6–24.
Figure Legends
Fig. 1 Classic-type EWS/pPNET: core biopsy (A and B) and relative excision specimen (C-F). A, Low magnification of fragments from a SRBCT (H&E, original magnification ×80). B, Serial section showing diffuse nuclear expression of cyclin D1 (immunoperoxidase, ×80; [AU: inset
ACCEPTED MANUSCRIPT magnification?]). C, H&E staining showing a SRBCT with hemorrhagic areas and pseudocystic spaces (×60). D, Serial section showing diffuse immunostaining for cyclin D1 (×60). E, High
T
magnification showing diffuse nuclear staining of cyclin D1 by neoplastic cells (×200). F, All
RI P
neoplastic cells show diffuse cell membranous staining for CD99, the conventional marker of
SC
EWS/pPNET (×200).
Fig. 2 Poorly differentiated neuroblastoma, incisional biopsy. Serial sections of low (A-C) and high
MA NU
(D-F) magnifications stained with H&E (A and D), NB-84 (B and E) and cyclin D1 (C and F). Arrows in panels A-C show localization of panels D-F. NB-84 and cyclin D1 are diffusely expressed in the cytoplasm and nuclei of neoplastic cells, respectively. (A-C, ×40; D-F, ×100; [AU:
ED
inset magnification?]).
PT
Fig. 3 Surgical specimens from poorly differentiated neuroblastoma (A-C) and intermixed ganglioneuroblastoma (D-F). A, H&E staining showing a SRBCT with nodular growth pattern and
CE
Homer-Wright pseudorosettes. B, The cytoplasm of neoplastic cells and Homer-Wright
AC
pseudorosettes are stained with NB84. C, Most undifferentiated neoplastic cells are stained with cyclin D1. D, H&E staining showing a typical intermixed ganglioneuroblastoma. E, Serial section showing that cyclin D1 expression is restricted to the neuroblastic component, while the surrounding ganglioneuromatous component is negative. F, Higher magnification: cyclin D1 stains the nuclei of undifferentiated neuroblasts, while developing ganglion cells are negative. A-C, ×100; D and E, ×80; F, ×200.
Fig. 4 Different histotypes of SRBCT lacking cyclin D1 immunostaining. Low magnification of serial sections stained, respectively, with H&E and cyclin D1, relative to embryonal rhabdomyosarcoma (A and B), solid variant alveolar rhabdomyosarcoma (C and ,D), lymphoblastic lymphoma (E and F), blastematous dominant WT (G and H) and desmoplastic small round cell
ACCEPTED MANUSCRIPT tumor (I and J). Cyclin D1 is negative with the exception of some endothelial cells of intratumoral blood vessels (D) and epithelial cells of WT (H, inset). A, C, E, G, I: H&E staining; A and B, E and
AC
CE
PT
ED
MA NU
SC
RI P
T
F, original magnifications ×80; C and D, ×100; G-J, ×60.
ACCEPTED MANUSCRIPT Table Immunohistochemical expression of cyclin D1 in SRBCTs (n = 128)
1+
0
0
0
2+
0
0
0
GN WT DSRCT LL RMS (n=5) (n=15) (n=6) (n=10) (n=26) 5/5 9/15b 3/6 10/10 26/26 (100%) (60%) (50%) (100%) (100%) 0 6/15(b) 3/6 0 0 (40%) (50%) 0 0 0 0 0
T
GNB (n=14) 0
RI P
Expression EWS/pPNET NB (n=30) (n=22) Negative 0 0
30/30 22/22 14/14a 0 0 0 0 0 (100%) (100%) (100%) NOTE. Negative, <1% positive cells; 1+, focal staining (1%–10% positive cells); 2+, heterogeneous staining (11%–50% positive cells); 3+, diffuse staining (>50% positive cells).
MA NU
SC
3+
Abbreviations: EWS/pPNET, Ewing sarcoma/primitive peripheral neuroectodermal tumor; NB, neuroblastoma; GNB, ganglioneuroblastoma; GN, ganglioneuroma; WT, Wilms’ tumor; DSRCT, desmoplastic small round cell tumor; LL, lymphoblastic lymphoma; RMS, rhabdomyosarcoma. Immunostaining restricted to neuroblastic component.
b
Immunostaining referred exclusively to blastematous component.
AC
CE
PT
ED
a
AC
CE
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
Figure 1
AC
CE
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
Figure 2
AC
CE
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
Figure 3
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 4
S0046-8177(16)30264-7 doi: 10.1016/j.humpath.2016.07.038 YHUPA 4038
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
22 June 2016 20 July 2016 22 July 2016
Please cite this article as: Magro Gaetano, Salvatorelli Lucia, Alaggio Rita, D’Agata Velia, Nicoletti Ferdinando, Di Cataldo Andrea, Parenti Rosalba, Diagnostic Utility of Cyclin D1 in the Diagnosis of Small Round Blue Cell Tumors in Children and Adolescents, Human Pathology (2016), doi: 10.1016/j.humpath.2016.07.038
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Diagnostic Utility of Cyclin D1 in the Diagnosis of Small Round Blue Cell Tumors in Children and Adolescents
T
Gaetano Magro MD, PhDa,*, Lucia Salvatorelli MDa, Rita Alaggio MDb, Velia D’Agatac,
RI P
Ferdinando Nicolettid, Andrea Di Cataldoe, Rosalba Parentif
a
SC
[AU: degrees?]
Department of Medical and Surgical Sciences and Advanced Technologies, G.F. Ingrassia,
MA NU
Azienda Ospedaliero-Universitaria “Policlinico-Vittorio Emanuele,” Anatomic Pathology Section, School of Medicine, University of Catania, Catania, Italy b
Department of Medicine-DIMED, Pathology Unit, University of Padova, Italy
c
Department of Biomedical and Biotechnological Sciences, Section of Human Anatomy and
ED
Histology, University of Catania, Catania, Italy. Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy
e
Pediatric Hematology and Oncology Unit, Department of Pediatrics, University of Catania
PT
d
Physiology Section, Department of Biomedical and Biotechnological Sciences, University of
AC
f
CE
Catania, Italy
Catania, Catania, Italy [AU: postal codes?]
*Address for correspondence: Prof. Gaetano Magro MD, PhD Department of Medical and Surgical Sciences and Advanced Technologies, G.F. Ingrassia, Azienda Ospedaliero-Universitaria “Policlinico-Vittorio Emanuele,” Anatomic Pathology Section, School of Medicine, University of Catania, Via S. Sofia 87, 95123 Catania, Italy E-mail address: [email protected]
ACCEPTED MANUSCRIPT Short title: Cyclin D1 in small round blue cell tumors Disclosures: The authors have no conflicts of interest or funding disclosures.
T
Keywords: Cyclin D1; Immunohistochemistry; Small round blue cell tumors; Children;
RI P
Adolescents
SC
Abstract Small round blue cell tumors (SRBCTs) of children and adolescents are often diagnostically challenging lesions. With the increasing diagnostic approach based on small biopsies,
MA NU
there is the need of specific immunomarkers that can help in the differential diagnosis among the different tumor histotypes to assure the patient a correct diagnosis for proper treatment. Based on our recent studies showing cyclin D1 overexpression in both Ewing’s sarcoma/primitive peripheral neuroectodermal tumor (EWS/pPNET) and peripheral neuroblastic tumors (neuroblastoma and
ED
ganglioneuroblastoma), we immunohistochemically assessed cyclin D1 immunoreactivity in 128
PT
cases of SRBCTs in children and adolescents in order to establish its potential utility in the differential diagnosis. All cases of EWS/pPNET and the undifferentiated/poorly differentiated
CE
neuroblastomatous component of all peripheral neuroblastic tumors exhibited strong and diffuse
AC
nuclear staining (>50% of neoplastic cells) for cyclin D1. In contrast, this marker was absent from rhabdomyosarcoma (regardless of subtype) and lymphoblastic lymphoma (either B- or T-cell precursor cells), while it was only focally detected (<5% of neoplastic cells) in some cases of Wilms tumor (blastemal component) and desmoplastic small round cell tumor. Our findings suggest that cyclin D1 can be exploitable as a diagnostic adjunct to conventional markers in confirming the diagnosis of EWS/pPNET or neuroblastoma/ganglioneuroblastoma. Its use in routine practice also may be helpful for those cases of SRBCT with undifferentiated morphology that are difficult to diagnose following application of the conventional markers.
ACCEPTED MANUSCRIPT 1. Introduction Small round blue cell tumors (SRBCTs) of children and adolescents are diagnostically
T
challenging lesions. Currently, their diagnosis is mostly based on morphology,
RI P
immunohistochemical analyses, as well as additional ancillary studies (cytogenetic and molecular studies). Diagnostic difficulties arise because some tumors show overlapping morphology and at
SC
least partly immunohistochemical features, or may present unusual clinical and/or pathological features [1-6]. As diagnosis based on small biopsies is increasing in daily practice, SRBCTs do
MA NU
represent a potential pitfall, especially if pathologists are not familiar with these entities [2,4,7-9]. Given the differences in disease biology, clinical behavior and therapeutic approach among the different histotypes of SRBCTs, an accurate diagnosis is mandatory for assuring the patient correct prognostic information and proper treatment. Accordingly, there is the need to identify novel
ED
specific immunomarkers that could be used successfully as cheap, fast and easily accessible
PT
confirmatory diagnostic tests in daily practice [2]. Cyclin D1 is a G1-specific cyclin that plays a crucial role in regulating cell cycle
CE
progression in G1/S transition, and therefore it is not at all surprising if it is overexpressed in
AC
several malignant tumors, including carcinomas, sarcomas and lymphomas [1,10]. Depending on tumor histotype, the altered expression of cyclin D1 is mainly due to different genomic alterations, including chromosomal translocation or amplification, post-transcriptional regulation or posttranslational protein stabilization [11-13]. Immunohistochemical expression of cyclin D1 has not been performed systematically in SRBCTs of children and adolescents. In this regard, an immunohistochemical study reported cyclin D1 expression only in 42% of cases of Ewing’s sarcoma/primitive peripheral neuroectodermal tumor (EWS/pPNET) [14], while another study, performed on peripheral neuroblastic tumors, found cyclin D1 overexpression in neuroblasts, in contrast to low expression in all cell types of ganglioneuromas [15]. Recently, we have found that cyclin D1 is overexpressed by soft tissue EWS/pPNET in children and adolescents, while it is absent from rhabdomyosarcoma, despite the subtype (embryonal, spindle cell/sclerosing, alveolar
ACCEPTED MANUSCRIPT subtypes) [16]. In another immunohistochemical study dealing with peripheral neuroblastic tumors of childhood, we showed that cyclin D1 expression was limited to the neuroblastic cell component
T
of neuroblastomas and ganglioneuroblastomas, whereas it is lacking, or only focally detectable, in
RI P
the maturing/mature ganglion cell component of differentiating neuroblastomas, ganglioneuroblastomas, and ganglioneuromas [17]. Notably, we also emphasize that cyclin D1 in
SC
neuroblastic tumors recapitulates its developmental expression [17]. These immunohistochemical results have been supported by publicly available oligonucleotide microarrays showing significant
MA NU
upregulation of cyclin D1 in both Ewing’s sarcoma and neuroblastoma as compared to normal tissues or other malignant tumors [18,19].
These promising results prompted us to evaluate the potential diagnostic utility of cyclin D1 as an immunomarker in the differential diagnosis of the most common tumors that usually exhibit
ED
small round cell morphology in children and adolescents. A large series of 128 cases of SRBCTs,
PT
including EWS/pPNET, rhabdomyosarcoma, Wilms tumor, lymphoblastic lymphoma and
CE
desmoplastic small round cell tumor, were immunohistochemically tested for cyclin D1 expression.
AC
2. Materials and methods 2.1. Ethics statement
The present research complied with the Helsinki Declaration and was approved by the Human Ethics Committee and the Research Ethics Committee of the University of Catania. All patients provided written informed consent according to institutional guidelines. Patients were informed that the resected specimens were stored by the sections of Anatomic Pathology of both Universities of Catania and Padova and might potentially be used for scientific research, and that their privacy would be maintained.
ACCEPTED MANUSCRIPT 2.2. Tissue specimens The cases were retrospectively retrieved from the surgical pathology archives of the section
T
of Anatomic Pathology, G.F. Ingrassia Department of Medical, Surgical, and Advanced
RI P
Technologies, at University of Catania and from the surgical pathology archives of the Italian reference center for pediatric soft tissue sarcomas at the University of Padova. Clinical data were
SC
obtained from the original pathology reports. Hematoxylin and eosin–stained slides (H&E) and a variable number of slides stained with several antibodies were available for each case. All the H&E
MA NU
slides were reviewed by two expert surgical pathologists (R.A. and G.M.) of the GIPOP (Italian Group of Oncologic Pediatric Pathology), and the diagnoses were histologically confirmed using the current well-established morphological criteria and immunohistochemical features [20-24]. Molecular data (fusion gene products) by reverse-transcription polymerase chain reaction (RT–
ED
PCR) were also available for some cases. At least one representative formalin-fixed, paraffin-
PT
embedded block was available for immunohistochemical analyses. We selected 30 cases of soft tissue EWS/pPNET, 26 cases of rhabdomyosarcoma, 41 cases
CE
of peripheral neuroblastic tumors, 15 cases of Wilms tumor (WT), 6 cases of desmoplastic small
AC
round cell tumor (DSRCT) and 10 cases of lymphoblastic lymphomas (pre-B or pre-T precursor cells). All tissue samples were derived from primary tumors of untreated patients. Most of the cases of EWS/pPNET, rhabdomyosarcoma and peripheral neuroblastic tumors were included in previously published studies [16,17]. Patients with EWS/pPNET diagnosis were 21 males and 9 females, with ages ranging from 4 to 19 years (median age 11.5 years). Tumors were from soft tissues of extremities (14 cases), chest wall (7 cases), paravertebral (5 cases), intra-abdominal (2 cases) and pelvic (1 case) regions, and retroperitoneum (1 case). Most tumors (26/30) were derived from incisional biopsies, with the exception of 4 cases (2 cases from surgically excised specimen; 2 cases from needle core biopsy). As CIC-DUX4 and BCOR fusion sarcomas with Ewing-like morphology are emerging entities [2529], we included in this study exclusively classic-type EWS/pPNET. Histologically, tumors were
ACCEPTED MANUSCRIPT composed of uniform small round blue cells arranged in a vaguely lobular to sheet-like growth pattern, and they were strongly and diffusely stained (along the cell membrane of neoplastic cells)
T
with CD99. None of the cases selected showed atypical morphology (heterogeneity in tumor cell
RI P
size and shape, nuclear enlargement, irregular nuclear contours, frequent prominent nucleoli, diffuse myxoid change, delicate arborizing capillary network) and/or ambiguous CD99
SC
immunostaining (focal, heterogeneous, or incomplete staining along the cell membrane). All cases were negative for desmin, myogenin, MyoD1, NB84, TdT, CD56, α-smooth muscle actin, pan-
MA NU
cytokeratins, S100 protein, and WT1. FLI-1 was positive in the cases that were originally tested for this antibody (15/15 cases). Molecular data were also available for 18 cases of EWS/pPNET. RT– PCR identified the EWS-FL1 fusion product.
The cases of rhabdomyosarcoma were represented by 10 cases of embryonal
ED
rhabdomyosarcoma, 2 cases of spindle cell/sclerosing rhabdomyosarcoma and 14 cases of alveolar
PT
rhabdomyosarcoma. Most tumors (18/26) derived from incisional biopsies, while the remaining cases (8/26) were from surgically excised specimens. The patients affected with embryonal
CE
rhabdomyosarcoma were 7 males and 3 females, with ages ranging from 1 to 11 years (median age
AC
6.5 years). Tumors occurred in the orbit (2 cases), palate (2 cases), bladder (2 cases), trunk (1 case), paratesticular (1 case), nasopharynx (1 case), auditory canal (1 case). The patients affected with spindle cell/sclerosing rhabdomyosarcoma were 2 males (9 and 11 years), and both tumors occurred in the palate. The patients affected with alveolar rhabdomyosarcoma were 7 males and 7 females, with ages ranging from 1 to 12 years (median age 9.7 years). Tumors occurred in soft tissues of abdominal wall (4 cases), extremities (3 cases), testis (3 cases), pelvic region (2 case), orbit (1 case) and groin (1 case). All cases of rhabdomyosarcomas, regardless of histological subtype, were positive (albeit with variable extension) for desmin, myogenin and MyoD1, while they were negative for CD99, NB84, CD56, FLI-1, TdT, WT1 (negative nuclear staining), α-smooth muscle actin, S-100 protein, and pan-cytokeratins. Molecular data were also available in 9 cases of alveolar
ACCEPTED MANUSCRIPT rhabdomyosarcoma. RT-PCR identified the PAX3/FKHR fusion product in 6 cases, whereas the other 3 cases tested were fusion negative.
T
The cases of peripheral neuroblastic tumors were classified based on well-established
RI P
morphological criteria [22,23]: (i) 22 Schwannian stroma-poor tumors (1 undifferentiated neuroblastoma, 19 poorly differentiated neuroblastomas, 2 differentiating neuroblastomas); (ii) 14
SC
Schwannian stroma-rich tumors (12 intermixed ganglioneuroblastomas, 2 nodular ganglioneuroblastomas); (iii) 5 Schwannian stroma-predominant tumors (5 mature
MA NU
ganglioneuromas). The patients were 23 males and 18 females, with ages ranging from 1 month to 9 years (median age 4.2 years). Tumors occurred in adrenal gland, retroperitoneum, superior and posterior mediastinum.
Patients with WT diagnosis were 12 females and 3 males, with ages ranging from 1 to 8
ED
years (median age 4.6 years). All cases but one derived from nephrectomy specimens.
PT
Histologically, 10 cases showed a triphasic pattern, while 4 cases were blastemal predominant (only focal epithelial and stromal components were identified). The single case of WT, in which only
CE
biopsy was available, showed exclusively a blastematous component. In all cases examined,
AC
anaplasia—namely, large pleomorphic cells with marked nuclear atypia and atypical (multipolar) mitotic figures—was not observed. Patients with DSRCT diagnosis were 4 males and 2 females, with ages ranging from 8 to 18 years. All tumors were from intra-abdominal locations and immunohistochemically coexpressed cytokeratins, vimentin, desmin, and WT1 (carboxy-terminus WT1 antibody). They were negative for myogenin, MyoD1, CD99, TdT, and NB84. Molecular data were also available in 4 cases. RT– PCR identified the EWSR-WT1 fusion transcript. Patients with lymphoblastic lymphomas (7 patients with B-precursor cell lymphomas; 3 patients with T-precursor cell lymphomas) were 8 males and 2 females, with ages ranging from 1 to 19 years (median age 6 years). Precursor T-cell lymphomas were located in the anterior mediastinum, while the precursor-B-cell lymphomas presented as enlarged cervical (2 cases) and
ACCEPTED MANUSCRIPT axillary (1 case) lymph nodes, testicular masses (2 cases), intra-abdominal mass (1 case) and palate mass (1 case).
T
Immunohistochemical analyses were performed as previously reported in detail [16,17].
RI P
Briefly, after appropriate deparaffinization and pretreatments, sections were incubated with anti– cyclin D1 (SP4, NeoMarkers [AU: supplier location?]; prediluted antibody) at pH 6.0 for 60 min at
SC
room temperature. Microwave pretreatment was crucial to enhance the staining in all examined samples. Accordingly, all sections were pretreated with citrate buffer (pH 6.0) and exposed to
MA NU
radiation in a microwave oven. To reduce the commonly seen nonspecific immunoreactivity due to endogenous biotin, sections were pretreated with 10 mg/mL of ovalbumin in phosphate-buffered saline (PBS) followed by 0.2% biotin in PBS, each for 15 min at room temperature. Bound antibody was revealed by incubation with 3,3ʹ-diaminobenzidine (Sigma–Aldrich, St. Louis MO, USA) in
ED
0.01% H2O2 for 5 min at room temperature. Sections were then counterstained with hematoxylin,
PT
dehydrated, and mounted. Negative controls, involving the omission of the primary antibody, were included. The percentage of positively stained cells was assessed by semiquantitative optical
CE
analysis according to a four-tiered system (<1% positive cells, negative staining; 1%–10% positive
AC
cells, focal staining; 11%–50% positive cells, heterogeneous staining; >50% positive cells, diffuse staining). Staining intensity was graded as weak, moderate, or strong intensity.
3. Results Immunohistochemical results are summarized in the Table. Among SRBCTs, only EWS/pPNET and neuroblastic tumors showed diffuse expression of cyclin D1. All cases (30/30) of EWS/pPNET exhibited a strong and diffuse nuclear immunoreactivity for cyclin D1 (>50% of stained neoplastic cells) regardless of the tissue specimen tested (incisional, core needle biopsy, surgical excision) (Fig. 1B and D). The percentage of positive cells varied in the different cases, ranging from 60% to 95%. As expected, cyclin D1 was exclusively confined to the nuclei of tumor cells, with no immunoreactivity in their cytoplasm (Fig. 1B, D, E).
ACCEPTED MANUSCRIPT As far as neuroblastic tumors are concerned, the expression of cyclin D1 varied among the different cell types. We observed a diffuse (>70% of neoplastic cells) cyclin D1 nuclear staining
T
restricted to undifferentiated or poorly differentiated neuroblastic cells, while it was absent or only
RI P
focally (<5% of neoplastic cells) found in the neuroblastic cells with clear-cut ganglion cell differentiation, as well as in the mature ganglion cells (Figs. 2 and 3). Similar results were obtained
SC
regardless of the tissue specimen tested (incisional, core needle biopsy, surgical excision). Briefly, the highest cyclin D1 expression was found in both undifferentiated/poorly differentiated
MA NU
neuroblastomas (Fig. 2C and F, Fig. 3C) and in the neuroblastic component of ganglioneuroblastomas (both intermixed and nodular types) (Fig. 3E and F). No cyclin D1 expression was detected in the Schwann cell component of both ganglioneuroblastomas and ganglioneuromas.
ED
The other SRBCTs were negative or only focally positive for cyclin D1. Notably, no case of
PT
rhabdomyosarcoma (0/26), regardless histologic subtypes (Fig. 4B and D), or lymphoblastic lymphoma (0/10) (Fig. 4F) showed cyclin D1 immunoreactivity. Blastemal areas of WTs were
CE
negative (9/15 cases) or only focally (5%-10% stained cells) positive (6/15 cases) for cyclin D1
AC
(Fig. 4H). Similarly, 3 cases of DSRCT were negative (Fig. 4L), while the remaining 3 cases exhibited focal immunoreactivity (5%-10% stained cells). In all cases of SRBCTs tested, cyclin D1 variably stained the nuclei of endothelial cells of intratumoral blood vessels and served as internal control (Fig. 4D).
4. Discussion The differential diagnosis of SRBCTs can be challenging, especially in some clinicopathologic contexts. This is particularly true when SRBCTs are located in the abdominal cavity, because they can present overlapping clinicopathologic features. In daily practice, it is not rare that radiological imaging is not conclusive in identifying the organ (adrenal vs kidney; kidney vs retroperitoneum) where a tumor mass arises in children or adolescents. In this regard, a WT may
ACCEPTED MANUSCRIPT be so large in size that it could be difficult to establish with certainty its origin from the kidney. In addition, there is the possibility that other SRBCTs, such as EWS/pPNET or DSRCT, may
T
primarily arise in visceral organs, including kidney and retroperitoneum [3,24,30]. Therefore,
RI P
distinguishing a blastemal-predominant WT from other SRBCTs may be extremely difficult, especially if the pathologist is evaluating small biopsies from a renal/intra-abdominal mass. In
SC
addition, the presence of epithelial elements in the context of an undifferentiated cellular component, although highly suggestive, is not diagnostic of WT, as some cases of DSRCT may
MA NU
exhibit glandular differentiation. Similarly diagnostic problems may arise if bioptic material is obtained from an undifferentiated or poorly differentiated neuroblastoma located in the abdominal cavity. In fact, the presence of small round blue cells as the only cytotype, together with the absence of neuropil, makes very difficult the morphological distinction of undifferentiated neuroblastoma
ED
from other SRBCTs, especially EWS/pPNET and lymphoblastic lymphoma. Based on these
PT
considerations, the availability of highly sensitive and specific immunomarkers for the different tumor histotypes is crucial for an accurate diagnosis of the SRBCTs in children and adolescents.
CE
Our preliminary results showed that cyclin D1 is overexpressed in both soft tissue
AC
EWS/pPNET and neuroblastomas/ganglioneuroblastomas of children and adolescents [16,17]. The goal of the present study was to validate these results by assessing the potential diagnostic utility of cyclin D1 in the differential diagnosis of the most common SRBCTs. We showed that all cases (30/30) of soft tissue EWS/pPNET and the undifferentiated/poorly differentiated neuroblastomatous component of peripheral neuroblastic tumors exhibited a strong and diffuse (>50% of stained neoplastic cells) nuclear staining for cyclin D1. In contrast, this marker was absent from rhabdomyosarcoma (regardless of the subtype) and lymphoblastic lymphoma (either B- or T-cell precursor cells). Cyclin D1 immunoreactivity was also absent, or only focally detected (<5% of neoplastic cells), in the blastemal component of WT and in the neoplastic cells of DSRCT. Two recent studies on pediatric renal tumors obtained similar results, with absent to focal expression of
ACCEPTED MANUSCRIPT cyclin D1 in a small series of WT [31,32]. Our results suggest that cyclin D1 is a useful marker in separating EWS/pPNET and neuroblastomas/ganglioneuroblastomas from other SRBCTs.
T
As the immunohistochemical expression of cyclin D1 has been documented in a variety of
RI P
malignant tumors [33-38], its use as a single diagnostic immunomarker should be avoided. However, we suggest that a diffuse nuclear expression of cyclin D1, in the appropriate
SC
clinicopathologic context, can be exploitable as a diagnostic adjunct to conventional markers, such as CD99 and FLI-1 or NB84 and CD56, in confirming the diagnosis of EWS/pPNET or
MA NU
neuroblastoma/ganglioneuroblastoma, respectively. In addition cyclin D1 immunoreactivity may have an informative value when dealing with a SRBCT exhibiting undifferentiated morphology, which is difficult to diagnose even following application of the conventional markers (ambiguous staining). Diffuse and strong cyclin D1 expression should prompt pathologists to perform genetic
ED
and/or molecular studies as confirmatory tests for EWS/pPNET or
PT
neuroblastomas/ganglioneuroblastomas.
Although the main purpose of this study was to evaluate the use of cyclin D1 in the
CE
differential diagnosis of SRBCTs in children and adolescents, its diffuse expression either in
AC
EWS/pPNETs or neuroblastomas/ganglioneuroblastoma provides the rationale for exploiting cyclin D1 as a target for new strategic therapies. In this regard, there are promising phase I and II clinical trials based on cyclin D1 overexpression [11]. The mechanisms proposed range from cyclin D1 downregulation to inhibition of cyclin D1 action. The most immediately feasible approach is directed to inhibition of the cyclin D1–CDK4/CDK6 complex to induce a decrease of cell proliferation. In several emerging studies, cyclin D1 is the primary target of compounds such as cytotoxics, radiotherapy, and mTOR inhibitors able to determine the degradation of overexpressing cyclin D1 cancer cells [12]. Another proposed mechanism is the use of competitive inhibitors for specific domains in cyclin D1 or by substrate mimetics [12]. To date, the use of only one of these methods has had limited success; thus the trend would be the use of combined therapies, for
ACCEPTED MANUSCRIPT example, the most promising combination is among the inhibitors of CDKs and cytotoxic drugs
T
[12].
RI P
Acknowledgments
The authors thank Dr.ssa Spoto Graziana for her excellent technical assistance. The paper was
SC
supported by the Italian funding FIR2014.
MA NU
References
[1] Coffin CM, Alaggio R, Dehner LP. Some general considerations about the clinicopathologic aspects of soft tissue tumors in children and adolescents. Pediatr Dev Pathol 2012;15:11-25. [2] Magro G, Longo FR, Angelico G, Spadola S, Amore FF, Salvatorelli L. Immunohistochemistry
ED
as potential diagnostic pitfall in the most common solid tumors of children and adolescents.
PT
Acta Histochem 2015;117:397-414.
[3] Arnold MA, Schoenfield L, Limketkai BN, Arnold CA. Diagnostic pitfalls of differentiating
CE
desmoplastic small round cell tumor (DSRCT) from Wilms tumor (WT): overlapping
AC
morphologic and immunohistochemical features. Am J Surg Pathol 2014;38:1220-6. [4] Alaggio R, Boldrini R, Di Venosa B, Rosolen A, Bisogno G, Magro G. Pediatric extra-renal rhabdoid tumors with unusual morphology: a diagnostic pitfall for small biopsies. Pathol Res Pract 2009;205:451-7. [5] Magro G, Greco P, Alaggio R, Gangemi P, Ninfo V. Polypoid angiomyofibroblastoma-like tumor of the oral cavity: a hitherto unreported soft tissue tumor mimicking embryonal rhabdomyosarcoma. Pathol Res Pract 2008;204:837-43. [6] Bahrami A, Gown AM, Baird GS, Hicks MJ, Folpe AL. Aberrant expression of epithelial and neuroendocrine markers in alveolar rhabdomyosarcoma: a potentially serious diagnostic pitfall. Mod Pathol 2008;21:795–806.
ACCEPTED MANUSCRIPT [7] Wang H, Li F, Liu J, Zhang S. Ultrasound-guided core needle biopsy in diagnosis of abdominal and pelvic neoplasm in pediatric patients. Pediatr Surg Int 2014;30:31-7.
T
[8] Gustafson S, Medeiros LJ, Kalhor N, Bueso-Ramos CE. Anaplastic large cell lymphoma:
RI P
another entity in the differential diagnosis of small round blue cell tumors. Ann Diagn Pathol 2009;13:413-27.
SC
[9] Fowler DJ, Malone M, Chisholm J, Roebuck D, Sebire NJ. Primary thoracic myxoid variant of extrarenal rhabdoid tumor in childhood. Fetal Pediatr Pathol 2006;25:159-68.
MA NU
[10] Zukerberg LR, Yang WI, Arnold A, Harris NL. Cyclin D1 expression in non-Hodgkin's lymphomas. Detection by immunohistochemistry. Am J Clin Pathol 1995;103:756-60. [11] Musgrove EA, Caldon CE, Barraclough J, Stone A, Sutherland RL. Cyclin D as a therapeutic target in cancer. Nat Rev Cancer 2011;11:558-72.
ED
[12] Kim JK, Diehl JA. Nuclear cyclin D1: an oncogenic driver in human cancer. J Cell Physiol
PT
2009; 220:292-6.
[13] Pestell RG. New roles of cyclin D1. Am J Pathol 2013;183:3-9.
CE
[14] Fuchs B, Inwards CY, Janknecht R. Vascular endothelial growth factor expression is up-
AC
regulated by EWS-ETS oncoproteins and Sp1 and may represent an independent predictor of survival in Ewing’s sarcoma. Clin Cancer Res 2004;10:1344-53. [15] Molenaar JJ, Ebus ME, Koster J, et al. Cyclin D1 and CDK4 activity contribute to the undifferentiated phenotype in neuroblastoma. Cancer Res 2008;68:2599-609. [16] Magro G, Brancato F, Musumeci G, Alaggio R, Parenti R, Salvatorelli L. Cyclin D1 is a useful marker for soft tissue Ewing’s sarcoma/peripheral primitive neuroectodermal tumor in children and adolescents: a comparative immunohistochemical study with rhabdomyosarcoma. Acta Histochem 2015;117:460-7. [17] Magro G, Salvatorelli L, Di Cataldo A, Musumeci G, Spoto G, Parenti R. Cyclin D1 in human neuroblastic tumors recapitulates its developmental expression: An immunohistochemical study. Acta Histochem 2015;117:415-24.
ACCEPTED MANUSCRIPT [18] Fagone P, Nicoletti F, Vecchio GM, Parenti R, Magro G. Cyclin D1 in pediatric neuroblastic tumors: a microarray analysis. Acta Histochem 2015;117:820-3.
T
[19] Fagone P, Nicoletti F, Salvatorelli L, Musumeci G, Magro G. Cyclin D1 and Ewing’s
RI P
sarcoma/PNET: A microarray analysis. Acta Histochem 2015;117:824-8. [20] Parham DM, Alaggio R, Coffin CM. Myogenic tumors in children and adolescents. Pediatr
SC
Dev Pathol 2012;15:211-38.
[21] Tsokos M, Alaggio RD, Dehner LP, Dickman PS. Ewing sarcoma/peripheral primitive
MA NU
neuroectodermal tumor and related tumors. Pediatr Dev Pathol 2012;15:108-26 [22] Joshy VV, Silverman JF. Pathology of neuroblastic tumors. Semin Diagn Pathol 1994;11:107– 11.
[23] Shimada H, Ambros IM, Dehner LP, et al. The International Neuroblastoma Pathology
ED
Classification (the Shimada system). Cancer 1999;86:364-72.
PT
[24] da Silva RC, Medeiros Filho P, Chioato L, Silva TR, Ribeiro SM, Bacchi CE. Desmoplastic small round cell tumor of the kidney mimicking Wilms tumor: a case report and review of the
CE
literature. Appl Immunohistochem Mol Morphol 2009;17:557-62.
AC
[25] Graham C, Chilton-MacNeill S, Zielenska M, Somers GR. The CIC-DUX4 fusion transcript is present in a subgroup of pediatric primitive round cell sarcomas. HUM PATHOL 2012;43:180-9. [26] Italiano A, Sung YS, Zhang L, et al. High prevalence of CIC fusion with double-homeobox (DUX4) transcription factors in EWSR1-negative undifferentiated small blue round cell sarcomas. Genes Chromosomes Cancer 2012;51:207-18. [27] Antonescu C. Round cell sarcomas beyond Ewing: emerging entities. Histopathology 2014;64:26-37. [28] Machado I, Navarro S, Llombart-Bosch A. Ewing sarcoma and the new emerging Ewing-like sarcomas: (CIC and BCOR-rearranged-sarcomas). A systematic review. Histol Histopathol. 2016;31:1169-91.
ACCEPTED MANUSCRIPT [29] Kao YC, Sung YS, Zhang L, et al. Recurrent BCOR internal tandem duplication and YWHAENUTM2B fusions in soft tissue undifferentiated round cell sarcoma of infancy: overlapping
T
genetic features with clear cell sarcoma of kidney. Am J Surg Pathol 2016;40:1009-20.
RI P
[30] Wang LL, Perlman EJ, Vujanic GM, et al. Desmoplastic small round cell tumor of the kidney in childhood. Am J Surg Pathol 2007;31:576-84.
SC
[31] Jet Aw S, Hong Kuick C, Hwee Yong M, et al. Novel karyotypes and cyclin D1 immunoreactivity in clear cell sarcoma of the kidney. Pediatr Dev Pathol 2015;18:297-304.
MA NU
[32] Mirkovic J, Calicchio M, Fletcher CD, Perez-Atayde AR. Diffuse and strong cyclin D1 immunoreactivity in clear cell sarcoma of the kidney. Histopathology 2015;67:306-12. [33] Diehl JA. Cycling to cancer with cyclin D1. Cancer Biol Ther 2002;1:226–31. [34] Fu M, Wang C, Li Z, Sakamaki T, Pestell RG. Minireview cyclin D1: normal and abnormal
ED
functions. Endocrinology 2004;145:5439–47.
PT
[35] Sutherland RL, Musgrove EA. Cyclins and breast cancer. J Mammary Gland Biol Neoplasia 2004;9:95–104.
CE
[36] Knudsen KE, Diehl JA, Haiman CA, Knudsen ES. Cyclin D1: polymorphism, aberrant splicing
AC
and cancer risk. Oncogene 2006;25:1620–8. [37] Musgrove EA. Cyclins: roles in mitogenic signaling and oncogenic transformation. Growth Factors 2006;24:13–9. [38] Alao JP. The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention. Mol Cancer 2007;2:6–24.
Figure Legends
Fig. 1 Classic-type EWS/pPNET: core biopsy (A and B) and relative excision specimen (C-F). A, Low magnification of fragments from a SRBCT (H&E, original magnification ×80). B, Serial section showing diffuse nuclear expression of cyclin D1 (immunoperoxidase, ×80; [AU: inset
ACCEPTED MANUSCRIPT magnification?]). C, H&E staining showing a SRBCT with hemorrhagic areas and pseudocystic spaces (×60). D, Serial section showing diffuse immunostaining for cyclin D1 (×60). E, High
T
magnification showing diffuse nuclear staining of cyclin D1 by neoplastic cells (×200). F, All
RI P
neoplastic cells show diffuse cell membranous staining for CD99, the conventional marker of
SC
EWS/pPNET (×200).
Fig. 2 Poorly differentiated neuroblastoma, incisional biopsy. Serial sections of low (A-C) and high
MA NU
(D-F) magnifications stained with H&E (A and D), NB-84 (B and E) and cyclin D1 (C and F). Arrows in panels A-C show localization of panels D-F. NB-84 and cyclin D1 are diffusely expressed in the cytoplasm and nuclei of neoplastic cells, respectively. (A-C, ×40; D-F, ×100; [AU:
ED
inset magnification?]).
PT
Fig. 3 Surgical specimens from poorly differentiated neuroblastoma (A-C) and intermixed ganglioneuroblastoma (D-F). A, H&E staining showing a SRBCT with nodular growth pattern and
CE
Homer-Wright pseudorosettes. B, The cytoplasm of neoplastic cells and Homer-Wright
AC
pseudorosettes are stained with NB84. C, Most undifferentiated neoplastic cells are stained with cyclin D1. D, H&E staining showing a typical intermixed ganglioneuroblastoma. E, Serial section showing that cyclin D1 expression is restricted to the neuroblastic component, while the surrounding ganglioneuromatous component is negative. F, Higher magnification: cyclin D1 stains the nuclei of undifferentiated neuroblasts, while developing ganglion cells are negative. A-C, ×100; D and E, ×80; F, ×200.
Fig. 4 Different histotypes of SRBCT lacking cyclin D1 immunostaining. Low magnification of serial sections stained, respectively, with H&E and cyclin D1, relative to embryonal rhabdomyosarcoma (A and B), solid variant alveolar rhabdomyosarcoma (C and ,D), lymphoblastic lymphoma (E and F), blastematous dominant WT (G and H) and desmoplastic small round cell
ACCEPTED MANUSCRIPT tumor (I and J). Cyclin D1 is negative with the exception of some endothelial cells of intratumoral blood vessels (D) and epithelial cells of WT (H, inset). A, C, E, G, I: H&E staining; A and B, E and
AC
CE
PT
ED
MA NU
SC
RI P
T
F, original magnifications ×80; C and D, ×100; G-J, ×60.
ACCEPTED MANUSCRIPT Table Immunohistochemical expression of cyclin D1 in SRBCTs (n = 128)
1+
0
0
0
2+
0
0
0
GN WT DSRCT LL RMS (n=5) (n=15) (n=6) (n=10) (n=26) 5/5 9/15b 3/6 10/10 26/26 (100%) (60%) (50%) (100%) (100%) 0 6/15(b) 3/6 0 0 (40%) (50%) 0 0 0 0 0
T
GNB (n=14) 0
RI P
Expression EWS/pPNET NB (n=30) (n=22) Negative 0 0
30/30 22/22 14/14a 0 0 0 0 0 (100%) (100%) (100%) NOTE. Negative, <1% positive cells; 1+, focal staining (1%–10% positive cells); 2+, heterogeneous staining (11%–50% positive cells); 3+, diffuse staining (>50% positive cells).
MA NU
SC
3+
Abbreviations: EWS/pPNET, Ewing sarcoma/primitive peripheral neuroectodermal tumor; NB, neuroblastoma; GNB, ganglioneuroblastoma; GN, ganglioneuroma; WT, Wilms’ tumor; DSRCT, desmoplastic small round cell tumor; LL, lymphoblastic lymphoma; RMS, rhabdomyosarcoma. Immunostaining restricted to neuroblastic component.
b
Immunostaining referred exclusively to blastematous component.
AC
CE
PT
ED
a
AC
CE
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
Figure 1
AC
CE
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
Figure 2
AC
CE
PT
ED
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
Figure 3
MA NU
SC
RI P
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Figure 4