Accepted Manuscript

Calcifying Nested Stromal-Epithelial Tumor (CNSET) of the liver in Beckwith- Wiedemann syndrome

EUROPEAN JOURNAL OF MEDICAL GENETICS

Nasim Khoshnam, Haynes Robinson, Michael R. Clay, Lauren R. Schaffer, Scott E. Gillespie, Bahig M. Shehata

PII:

S1769-7212(16)30098-2

DOI:

10.1016/j.ejmg.2016.12.001

Reference:

EJMG 3237

To appear in:

European Journal of Medical Genetics

Received Date: 24 May 2016

Revised Date: 23 November 2016

Accepted Date: 1 December 2016

Please cite this article as: N. Khoshnam, H. Robinson, M.R. Clay, L.R. Schaffer, S.E. Gillespie, B.M. Shehata, Calcifying Nested Stromal-Epithelial Tumor (CNSET) of the liver in Beckwith-Wiedemann syndrome, European Journal of Medical Genetics (2017), doi: 10.1016/j.ejmg.2016.12.001.

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Calcifying Nested Stromal-Epithelial Tumor (CNSET) of the Liver in Beckwith- Wiedemann Syndrome

Running title: CNSET and BWS: A Newly Unrecognized Association Key Words: Calcifying Nested Stromal-Epithelial tumor (CNSET), Beckwith-Wiedemann Syndrome (BWS), Uniparental Disomy, Abnormal Methylation at 11p15.5

Nasim Khoshnam1, Haynes Robinson2, Michael R. Clay’, Lauren R. Schaffer’, Scott E Gillespie1, Bahig M. Shehata”.

Emory University School of Medicine, Department of Pathology, Atlanta, GA 30332 USA

Akron’s Children’s Hospital, Department of Medical Genetics, Akron, OH 44308 USA

Author of Correspondence;

Bahig M. Shehata, MD Professor of Pathology and Pediatrics Emory University School of Medicine 1405 Clifton Road, Atlanta, GA 30322-1101 Tel. 770-330-8282 Email: bshehat@emory.edu

ACCEPTÉ

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ABSTRACT

Calcifying nested stromal-epithelial tumor (CNSET) is a rare neoplasm. In the 31 reported cases, CNSET is predominantly found in young girls and women. Beckwith-Wiedemann syndrome (BWS) (OMIM #130650) is an overgrowth syndrome with an increased risk to develop cancer. Associations have been seen between BWS and embryonal tumors, especially Wilms tumor, hepatoblastoma, and adrenocortical carcinoma. Here we report on a female patient with BWS who presented with CNSET. Two other cases with the same association have been reported, with our case representing the third such reported in the literature. Although we recognize a potential reporting bias we speculate that CNSET may represent an unrecognized addition to the spectrum of BWS tumorigenesis.

INTRODUCTION

Calcifying nested stromal-epithelial tumor (CNSET) is a rare primary hepatic tumor characterized by a nested morphologic growth pattern composed of spindle and epithelioid cells with variable degrees of calcification and/or ossification. This non-hepatic, non-biliary primary

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liver neoplasm was first described by Ishak in 2001 (1) To our knowledge, only 31 cases were previously reported in the literature under a variety of names including: ossifying stromal- epithelial tumor, desmoplastic nested spindle cell tumor of the liver, and ossifying malignant mixed epithelial and stromal tumor (2) Predominantly found in young girls and women, CNSET is most commonly found in the right hemiliver (3).

Beckwith-Wiedemann syndrome (BWS) is a well-known overgrowth syndrome characterized by congenital malformations and tumor predisposition. It has since proven to display a phenotypic spectrum that includes varied prenatal and neonatal characteristics, a variety of structural anomalies, a range of altered growth potentials and an increased risk for benign and malignant neoplasia (4,5). The broadening phenotypic concept of BWS has been explained by recent understanding of the spectrum of different molecular genetic basis of BWS. It is now possible to show correlations between the four primary (epi)genetic mechanisms for the syndrome and the various phenotypic expressions of the syndrome. Similarly, it is also possible to correlate the frequency of specific tumor types with these four (epi)genetic mechanisms (6). These four principal (epi) genetic mechanisms are: (1) Loss of methylation of imprinting center 2, (2) Gain of methylation of imprinting center 1, (3) 11p15 paternal uniparental disomy and (4) variants of the CDKNIC gene (4,5,7). Children with BWS are at an increased risk of tumors, however the incidence varies depending on the genetic subgroup. The accumulative incidence of Wilms tumor is 37.6%, followed by hepatoblastoma (4.5%), and neuroblastoma (5.2%) (6).

Here we report a 14-year-old female with genetically confirmed BWS who presented with CNSET. To our knowledge, this is the third case of CNSET in a patient with BWS. Maloway et al. describe one case of a patient with CNSET concurrent with confirmed BWS in 2013, and another case is by Heerema-Mckenney et al. of a patient presenting with clinical

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diagnosis of BWS (8,9) We discuss the (epi)genetic relationship between CNSET and BWS, proposing that this may represent a previously unrecognized association.

CASE REPORT

A premature female patient (26 weeks) presented with Cushing syndrome, abdominal swelling and pain. The 14-year-old had a clinical diagnosis of BWS at age 3, presenting with macroglossia, macrognathia, mandibular prognathism, LV hypertrophy, and hypothyroidism. The diagnosis of BWS was then confirmed by molecular genetic analysis at age 13. Genetic diagnosis confirmed alterations in DNA methylation at two differentially methylated regions (DMRs) on the chromosome 11p15 showing loss of methylation at DMR2 (LOM-IC2) and normal methylation at DMR1. No deletions or duplications on 11p15were detected.

Investigation of her abdominal pain and swelling utilizing CT scan, showed an incidental finding of a large mass occupying the right lobe of the liver. Blood work showed elevated ACTH levels explaining the Cushingoid symptoms. Needle biopsy was performed. Histologically the tumor was composed of nests of spindled to epithelioid shaped cells with abundant pale to brightly eosinophilic, often vacuolated, cytoplasm. These nests were surrounded by cuffs of myofibroblastic stroma containing psammomatous calcifications, small foci of ossification, and a background bile ductular proliferation (Figure 1A-D). Immunohistochemical staining revealed the epithelioid cells to be positive for CD56, CD117, C-terminal WT1 (C-WT1, nuclear), N- terminal WT1 (N-WT1, cytoplasmic), cytokeratin AE1/AE3, and vimentin. The stromal cells were diffusely positive for SMA, N-WT1, C-WT1 (nuclear), and both populations were negative for HepPar-1 and Glypican-3 (Figure 2A-D). Additionally, Alpha Feta Protein (AFP) levels were

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within the normal range. The Ki-67 was <10%. The diagnosis of calcifying nested stromal- epithelial tumor (CNSET) was reached.

Chemotherapy was started without success and the patient underwent liver transplant. The resected specimen weighed 1780g, and showed an irregular encapsulated mass measuring 12.0 x 10.0 x 8.3 cm. The cut surface was whitish tan with central fibrosis. Additional sections from the resection showed large solid tumor nodules similar to the needle biopsy findings. A pseudo-capsule was identified and the surrounding liver parenchyma was unremarkable. Follow- up showed no recurrence of the tumor. Additionally, ACTH levels returned to normal and the Cushingoid appearance resolved.

DISCUSSION

Calcifying nested stromal-epithelial tumor (CNSET) of the liver was first described by Ishak et. al in 2001(1). These rare tumors are more frequent in young females (76%) with a mean age of onset at 15.2 years (10). The majority of the reported CNSET cases (55%) were discovered incidentally, and many of these were inaccurately reported under other tumor types within the differential diagnosis, principally hepatoblastoma, Wilms tumor, desmoplastic small round cell tumor (DSRCT), and synovial sarcoma. Although information on clinical outcomes is limited, the lesions do appear to have metastatic potential, with two cases of metastasis reported in the literature (9,11). As such, close post-operative follow-up is critical.

Beckwith-Wiedemann syndrome is an overgrowth syndrome that is characterized by an increased risk of cancer predisposition, specifically embryonal tumors, in children.

The non-neoplastic phenotypic characteristics of this patient are well compatible with those that correlate with LOM-IC2. Patients with LOM-IC2 have a very low risk for previously reported malignant tumors (4,6,7).Both our case and the case presented by Maloway et al. have been molecularly confirmed. Maloway et al. present a case of a girl delivered prematurely at 28 weeks’ gestation. The infant showed the facial characteristics of BWS . Her diagnosis of BWS was confirmed by documented hypermethylation of H19, so the mechanism was different compare to the mechanism in the present patient. Eventually, she developed nested stromal epithelial tumor (NSET) of the liver at age of 22 months (8). Heerema-Mckenney et al. published a series of six cases with CNSET. Patient 3 is a fourteen-year-old female who presented with omphalocele, renal abnormalities, intellectual disability and was clinically diagnosed as BWS. No genetic testing was performed to confirm this diagnosis (9), and the diagnosis BWS has to remain unconfirmed in this patient.

WT-1 positivity in the spindle component of the tumor has also been reported by Heerema-Mckenney et al. and Maloway et al., as in our case. Since none of the report cases of CNSET showed concurrent diagnosis of Wilms’ Tumor, the significance of this finding is most probably attributed to cross reaction between the WT-1 and BWS loci in the short arm of chromosome 11.

At present three cases of CNSET with concurrent BWS (of which two are molecularly confirmed) have been reported. This indicate that there is potentially a link between CNSET and BWS, although we recognize there may be a significant reporting bias, and numbers are still small. We conclude CNSET may occur more frequently in BWS patients than can be expected

7 Khoshnam et al. by chance. Studying large series of patients with CNSET and with BWS are needed to ensure this association is fortuitously or not.

HACCEPTED MANUS CRIPT

DISCLOSURE/CONFLICTS OF INTEREST

The authors of this manuscript have no conflicts of interest to declare.

ACCEPTED MANUS CRIPT

REFERENCES

1. Ishak K.G, Goodman Z.D, Stocker J.T, Miscellaneous malignant tumors (chapter 11). 2001. Tumors of the Liver and Intrahepatic Bile Ducts. Washington, DC: Armed Forces Institute of Pathology: 271-278.

2. Misra S, Bihari C. Desmoplastic nested spindle cell tumours and nested stromal epithelial tumours of the liver. 2016. APMIS 124:245-251.

3. Schaffer L.R, Shehata B.M, Yin J, Schemankewitz E, Alazraki A. Calcifying Nested stromal- epithelial tumor (CNSET) of the liver: a newly recognized entity to be considered in the radiologist’s differential diagnosis. 2015. Clin Imag 40(1): 137-139.

4. Mussa A, Russo S, de Crescenzo A, Freschi A, Calzari L, Maitz S, Macchiaiolo M, Molinatto C, Baldassarre G, Mariani M, Tarani L, Silengo M.C., Larizza L, Riccio A, Ferrero G.B. Fetal growth patterns in Beckwith-Wiedemann syndrome. 2016. Clin Genet 90: 21-27.

5. Shuman C, Beckwith J.B, Smith A.C, Weksberg R. Beckwith-Wiedemann Syndrome. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJH, Bird TD, Fong CT, Mefford HC, Smith RJH, Stephens K, editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2016.

6. Maas S.M, Vansenne F, Kadouch D.J.M, Ibrahim A, Bliek J, Hopman S, Mannens M.M, Merks J.H.M, Maher E.R, Hennekam R.C. Phenotype, Cancer Risk, and Surveillance in Beckwith- Wiedemann Syndrome Depending on Molecular Genetic Subgroups. 2016. Am J Med Genet Part A 170A: 2248-2260.

7. Mussa A, Peruzzi L, Chiesa N, De Crescenzo A, Russo S, Melis D, Tarami L, Baldassarre G, Larizza L, Riccio A, Silengo M, Ferrero G.B. Nephrological findings and genotype-phenotype correlation in Beckwith-Wiedemann syndrome. 2011. Pediatric Nephrol 27: 397-406.

8. Malowany J.I, Merritt N.H, Chan N.G, Ngan B. Nested Stromal Epithelial Tumor of the Liver in Beckwith-Wiedemann Syndrome. 2013. Pediatr Developm Pathol 16(4): 312-317.

9. Heerema-McKenney A, Leuschner I, Smith N, Sennesh J, Finegold MJ. Nested Stromal Epithelial Tumor of the Liver Six Cases of a Distincitve Pediatric Neoplasm With Frequent Calcifications and Association With Cushing Syndrome. 2005. Am J Surg Pathol 29(1): 10-20.

10. Wang Y, Zhou J, Huang W, Rao Q, Ma H, Zhou X. Calcifying Nested Stromal-Epithelial Tumor of the Liver: A Case Report and Review of Literature. 2015. Internat J Surg Pathol 19(2): 268- 272.

11. Maklouf H.R., Abdul-Al H, Wang G, Goodman Z.D. Calcifying Nested Stromal-epithelial Tumors of the Liver A Clinicopathologic, Immunohistochemical, and Molecular Genetic Study of 9 Cases with a Long-term Follow-up. 2009. Am J Surg Pathol 33(7): 976-983.

Figure 1: CNSET morphologic patterns

A. dense myofibroblastic stroma surrounding epithelioid nests

B. myofibroblastic stroma with reactive bile ductular proliferation

C. psammomatous calcifications

Figure 2: CNSET Immunohistochemical Staining

A. SMA showing dense staining of myofibroblastic stroma

C. C-Terminal WT-1 staining, diffuse nuclear staining

D. Higher magnification of C-terminal WT-1 staining.

FIGURE LEGEND D. heterotopic ossification Tal B. Negative HepPar-1 staining

ACCEPTED MANUS CRIPT

Table 1. CNSET finding in three patients with BWS.
Authors	Phenotype	Genotype	Tumor Histology
Maloway et	Exophthalmos,	Hypermethylation of H19	Numerous well-
al. (2013)	infraorbital creases,		demarcated spindle cell
	posterior helical pits, earlobe creases, small and		nodules separated by a thin collar of
	widely spaced teeth, full lips, macroglossia, prematurity, no		myofibroblasts and
			sclerotic tissue. Strong cytoplasmic cell
	organomegaly.		expression of vimentin and CD56. WT1-COOH in the spindle cell component.
Heerema-	Intestinal obstruction,	No genetic confirmation,	Areas of sheet-like
Mckeeney et	small left kidney,	diagnosis not confirmed	proliferation of mitotically
al.(2005)	intellectual		active cells, with necrosis
Patient #3	disabilityomphalocele,		and foamy histiocytes.
Patient #3	renal abnomalies		WT1-COOH in the spindle cell component.