Adamantinoma-Like Ewing Sarcoma of the Head and Neck: A Case-Series of a Rare and Challenging Diagnosis

Abstract

Adamantinoma-like Ewing sarcoma (ALES) is a rare malignant tumor characterized by EWSR1::FLI1 related fusions and complex epithelial differentiation. ALES poses a tremendous diagnostic challenge owing to its resemblance to a wide variety of common head and neck malignancies. We aimed to study the clinicopathologic spectrum of ALES diagnosed at our institute. A retrospective review of the clinical and pathologic features of all EWSR1-rearranged ALES cases was performed after confirming the diagnosis. The cases lacking EWSR1 rearrangement were excluded. A total of 7 patients were analyzed. The median age was 27 years (range 7–42 years). There were 4 males and 3 female patients. Tumors were distributed as follows: maxilla (n = 2), parotid (n = 2), nasal cavity (n = 1), ethmoid/maxilla (n = 1), and thyroid (n = 1). Tumor size ranged from 2.2 to 5.5 cm. On microscopy, tumors displayed nested-lobular architecture, monomorphic cells, and interlobular fibrotic stroma. Other features included: palisading (n = 5), squamous differentiation (n = 2), keratinization (n = 1), colonisation of salivary ducts (n = 1) and thyroid follicles (n = 1), follicle-like cysts (n = 3), calcification (n = 2), necrosis (n = 3). Mitotic rate was 4–15/2 mm². On immunohistochemistry, cytokeratins (100%), p40 (100%), strong/diffuse membranous CD99 (100%), NKX2.2 (100%), Fli-1 (71%), and synaptophysin (71%) was positive. Patients received chemotherapy (n = 7) and radiotherapy (n = 4). Two patients developed recurrence at 6 and 10 months; 3 developed metastases at 0, 6, and 25 months. ALES is a rare and aggressive malignancy that mimics diverse neoplasms common in the head and neck region. Awareness of the morphologic and immunohistochemistry spectrum of this tumor is essential to avoid diagnostic errors.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12105-022-01412-1.

Introduction

Adamantinoma-like Ewing sarcoma (ALES) is a rare malignant tumor characterized by EWSR1::FLI1 translocation and complex epithelial differentiation. First described by Bridge et al. [1], ALES is currently regarded as a variant of Ewing sarcoma on account of shared round cell morphology, CD99 (mic2) and NKX2.2 immunoreactivity, and the genetic hallmark of EWSR1::FLI1 fusion [2]. While up to 25–30% of classical Ewing sarcomas may exhibit epithelial differentiation, the keratin expression is usually limited and seen with low-molecular-weight cytokeratins [2]. In contradistinction, ALES displays diffuse immunoreactivity with p40/p63 and high-molecular-weight cytokeratins, with a subset exhibiting overt squamous differentiation and even keratin pearl formation, that quite set them apart from the classical Ewing sarcoma [3].

Initially described in the extremities, a striking proportion (74%) of ALES cases show a predilection for the head and neck region [3]. Nonetheless, ALES is a rare entity with only 28 head and neck cases reported to date [3–18]. While ALES diagnosis is challenging at all sites, occurrence in the head and neck poses additional difficulties as the presence of squamous differentiation or immunohistochemical epithelial reactivity can be mistaken for an epithelial malignancy. Owing to its rarity, little information is available on the clinical course as well as the optimal treatment of ALES, although reports hint at outcomes similar to or a slightly more favorable one than Ewing sarcoma after treatment with surgery, adjuvant chemotherapy, and radiotherapy [3]. Herein, we report seven cases of ALES arising in the head and neck and complement it with a review of the literature to underscore their distinguishing features and clinical behavior.

Materials and Methods

Clinical and pathologic data of all cases of ALES arising in the head and neck region were retrieved from the archives of the Department of Pathology at our tertiary-care oncology institute. The key words used for search were: ‘adamantinoma-like,’ ‘Ewing family of tumors’, ‘ALES’, ‘undifferentiated round cell sarcoma’, ‘Ewing sarcoma’, ‘atypical Ewing sarcoma’, ‘Ewing sarcoma with epithelial differentiation’. Hematoxylin & eosin-stained slides and immunohistochemistry (IHC) slides of all cases were reviewed and the diagnosis was reconfirmed [2].

Data on age, sex, symptomatology, tumor location, treatment details, local recurrence, metastasis, and the status at the last follow-up visit were recorded from the hospital’s electronic medical records. Pathologic features recorded were: tumor size, architectural pattern, cellular characteristics, epithelial differentiation (identified on IHC), squamous differentiation (morphologically recognizable as squamous cells), keratinization, stromal characteristics, mucosal surface involvement/ pagetoid spread, mitotic figure count (per 2 mm²), necrosis, lymphovascular invasion (LVI), perineural invasion (PNI), resection margins, lymph node (LN) status, and IHC profile.

Immunostaining was performed on a Benchmark XT autostainer (Ventana) using the MACH2 Ultraview polymer detection kit (Ventana) and included appropriate controls. The list of antibodies, clones, and dilutions used in the cases has been listed in the Supplementary table.

Fluorescence in-situ hybridization (FISH) for EWSR1 rearrangements was performed using LSI break apart, dual-color EWSR1 probe (Zytolight SPEC EWSR1 dual-color break-apart probe) consisting of two DNA probes: the first, a ~ 500 kb probe labeled in Spectrum Orange, flanking the 5` side of the EWSR1 gene; the second, a ~ 1100 kb probe labeled in Spectrum Green, flanking the 3` side of the EWSR1 gene. This probe kit can detect EWSR1 gene rearrangements involving the known breakpoints restricted to introns 7 through 10. The processed sections were stained with 4′-6-Diamidino-2-phenylindole (DAPI) and examined under a fluorescent microscope (Carl Zeiss, Axio Imager Z1, Germany), using AxioCam MRc5 camera and Axio vision Rel 4.5 software. The signals were manually scored and the tumor was considered positive if more than 15% of 100 cells analyzed showed a split signal/ break-apart. Molecular tests, by the FISH technique in all cases were reported by two authors (OS and BR).

Results

A total of ten patients with a diagnosis of head and neck ALES were identified. Out of these, two cases (sinonasal, and parotid) while showing malignant round cell morphology, strong and membranous CD99, diffuse AE1/AE3 and p40 positivity, were found to lack EWSR1 gene rearrangements on the FISH assay. For the third patient (peri-orbital), since the tissue was inadequate and the patient refused surgery, molecular confirmation was not possible. These three cases were excluded from the study. One patient with confirmed EWSR1 rearrangement using break-apart FISH testing done in an outside NABL-accredited laboratory was included. Hence, a total of 7 patients with EWSR1-rearranged ALES were included in the present study. Of these, six patients had been referred with a different initial diagnosis before presenting at our institute; these included: basaloid squamous cell carcinoma (SCC) (n = 2), sialoblastoma (n = 1), basal cell adenocarcinoma (n = 1), poorly differentiated thyroid carcinoma (n = 1); 1 patient (case 6) was diagnosed as solid pseudopapillary tumor of the pancreas (n = 1) on pancreatic biopsy. Diagnoses in these 6 cases were revised to ALES after review at our institute.

Clinical Findings

The details of the clinical findings of ALES patients are provided in Table 1. Briefly, the tumors were distributed in the sinonasal tract (n = 4), parotid (n = 2), and the thyroid (n = 1). The median age was 27 years with a male-to-female ratio of 1.3. The initial presentations were largely related to the tumor location: the patients with the parotid and thyroid tumors presented with a painless growing mass/swelling (n = 3) while the sinonasal tumors presented with epistaxis and nasal obstruction (n = 2), and persistent headaches (n = 1). Case 6 initially presented with recalcitrant abdominal pain and on evaluation was found to have disseminated malignancy with skeletal and pancreatic metastases. On examination, a tumor in the maxilla, presenting as an upper gingivobuccal proliferative mass, was found. Based on the mucosal involvement and the largest mass on imaging, the tumor was regarded as a maxillary primary in a multidisciplinary clinic. Six patients (except case 6) underwent surgical resection of their tumors.

Table 1.

Clinical features of Adamantinoma-like Ewing Sarcoma cases (n = 7)

Features	Case 1	Case 2	Case 3	Case 4	Case 5	Case 6	Case 7
Age	7 y/M	12 y/F	42/M	30 y/M	27 y/M	25/F	34/F
Site	Thyroid	Parotid	Parotid	Nasal cavity	Maxilla	Maxilla	Ethmoid/maxilla
Symptoms	Swelling	Mass	Swelling	Obstruction, epistaxis	Obstruction, pain	Abdominal pain	Headache
Surgery	Hemi thyroidectomy	Parotidectomy	Lt adequate parotidectomy	Rhinectomy  + premaxilla ectomy  + BL ND	Rt total Maxillectomy + SND	No	Ethmoidectomy
Radiotherapy	No	Yes (55.8 Gy/31# (@1.8 Gy/#)	No	Yes (45 Gy/ 25#/ 5 weeks)	Yes (55.8 Gy / 31 #)	Yes (Palliative RT for skeletal metastasis)	No
Chemotherapy	Yes (EFT 2001 protocol)	Yes (EFT 2001 protocol)	No (defaulted)	Yes (defaulted post RT; received VAC-IE 6 cycles at recurrence10 months later)	Yes (COG Induction protocol VAC/IE; consolidation with VAC/IE post RT)	Yes (EFT 2001)	Yes (VAC/IE)
Follow-up (duration)	32 months	26 months	NA	39 months	4 months	24 months	0
Recurrence	No	No	NA	Yes	Yes	No	No
Distant metastasis	Yes	No	NA	Yes	No	Yes	No
Status at last follow-up	AWD	ANED	NA	AWD (progressive disease)	ANED	AWD (progressive disease)	Ongoing treatment

M male, F female, Rt right, Lt left, BL ND bilateral neck dissection, SND selective neck dissection, RT radiotherapy, Gy Gray, COG Children Oncology Group, VAC/IE alternating vincristine + doxorubicin + cyclophosphamide and ifosfamide + etoposide, NA not available, AWD alive with disease, ANED alive with no evidence of disease

Pathologic Features

The pathologic characteristics of ALES are detailed in Table 2 and illustrated in Figs. 1, 2, 3, and 4. Briefly, the average tumor size was 3.9 cm (range, 2.2–5.5 cm). Macroscopy findings for sinonasal tumors were recorded as fleshy, homogenous, and grey-white. The thyroid tumor (case 1) was circumscribed nodular mass with a homogenous grey-white cut surface and lacking extrathyroidal spread. Both the parotid tumors were also grossly well-delineated and without extra-parenchymal spread.

Table 2.

Pathologic features of Adamantinoma-like Ewing Sarcoma cases (n = 7)

Features	Case 1	Case 2	Case 3	Case 4	Case 5	Case 6	Case 7
Site	Thyroid	Parotid	Parotid	Nasal cavity	Maxilla	Maxilla	Ethmoid/maxilla
Initial diagnosis	Poorly differentiated thyroid carcinoma	Sialoblastoma	Basal cell adenocarcinoma	Basaloid SCC	Basaloid SCC	Round cell tumor/solid pseudopapillary tumor	ALES
Tumor size (cms)	3.7	3.3	2.2	5	5.5	3.6*	4.3
Cell morphology	Round	Round	Basaloid	Round	Basaloid	Round	Round
Cellular appearance	Monotonous	Monotonous	Monotonous	Monotonous	Monotonous	Monotonous	Monotonous
Architecture	Nested	Sheets and nests	Nested-lobular	Nested-lobular, focally trabecular	Nested focally; sheets with fibrous bands	Nested	Nested
Rosettes	Focal	Absent	Absent	Absent	Focal	Absent	Absent
Palisading#	Focal	Focal	Focal	Present	Present	Absent	Absent
Squamous	Absent	Absent	Present; abrupt	Present; abrupt	Absent	Absent	Absent
Keratinization	Absent	Absent	Present	Absent	Absent	Absent	Absent
Calcification	Present	Absent	Present	Absent	Absent	Absent	Absent
Follicle-like spaces	Present	Present	Absent	Present	Absent	Absent	Absent
Stroma	Hyalinised	Minimal; thin fibrovascular septae	Fibrotic	Hyalinized	Fibrous bands; focally myxoid	Loose edematous	Hyalinized
Colonization	Present	Present	Absent	Absent	Absent	Absent	Absent
Mitotic rate/2mm2	4	5	15	12	7	12	6
Necrosis	Present; min	Absent	Present	Absent	Absent	Present	Absent
Lymphovascular invasion	Absent	Absent	Absent	Absent	Absent	Absent	Absent
Perineural invasion	Absent	Absent	Present	Absent	Absent	Absent	Absent
R status	R0	R0	R1	R0	R1	NA	R0
Underlying bone	NA	NA	NA	Cartilage involved	Involved	NA	Involved
Lymph nodes metastasis	Clinically node negative	Negative (0/3)	Negative (0/1)	Negative	Negative (0/29)	Not done	Clinically node negative

SCC squamous cell carcinoma, ALES Adamantinoma-like Ewing sarcoma, min minimal, R status resection margins status, R0 negative margins, R1 microscopically positive margins, NA not available

^*Tumor size on imaging

^# focal- < 50% of the tumor

Fig. 1.

ALES typically displayed lobules and nests of closely packed blue round cells separated by hyalinized stroma (A, B). Infrequently, diffuse cellular sheets with fewer nests were identified in the parotid (case 2; C). In the thyroid (case 1), the tumor revealed a nested architecture with a striking colonizing pattern of spread recognizable at low power (D)

Fig. 2.

ALES tumor cells were monomorphic with predominant round cell (A) or basaloid morphology (B). Peripheral palisading was conspicuous in some tumor islands; the arrow highlights abrupt squamous differentiation (C). Follicle-like spaces filled with pale eosinophilic fluid were identified within the tumor nests and sheets in the nasal (D) and the parotid (E) tumors. Solid nests of tumor cells were admixed with follicles displaying variably thick collars of neoplastic cells replacing normal thyroid follicular cells in a clinging pattern while retaining luminal (colloid) secretions (F)

Fig. 3.

Squamous nests, eddies as well as larger keratinized squamous pearls were identified centered abruptly within the monotonous round cells; black arrows highlight the squamous nests. Many of the keratinous pearls were calcified (A–D). Other features included colonization of thyroid follicles by tumor cells (E), pagetoid spread of tumor cells into the salivary ducts (F, G), entrapped ducts (H), and cracking artifacts separating large tumor nests (I)

Fig. 4.

Immunohistochemistry of ALES. The tumor cells were strongly and diffusely positive for AE1/AE3 (A), p40 (B), CD99 (C), and NKX2.2 (D). Fluorescent in situ hybridization revealed EWSR1 rearrangement (red-green ‘spilt’ signals in the inset, E E–F; DAPI × 1000)

On microscopy, while the tumors appeared relatively circumscribed at low power, the tumor edges revealed an infiltrative interface in all cases. At low magnification, all tumors displayed nests and lobules composed of closely packed blue round cells and separated by hyalinized stroma (Fig. 1). Higher magnification revealed round to epithelioid cells with fine chromatin, inconspicuous nucleoli, and negligible to scant pale cytoplasm that imparted a round cell (n = 4) or a basaloid (n = 3) appearance to the tumors (Fig. 2A, B). Nonetheless, tumor cells were conspicuous for their cellular monotony (Fig. 2A, B). Pseudo-rosettes were identified focally in 2 cases. At places, a few tumor nests exhibited peripheral palisading that was conspicuous in 2, focal in 3, and absent in 2 cases (Fig. 2C). While the nested architecture was characteristic, two cases revealed areas where nests coalesced to form solid sheets (Fig. 1C). Remarkably, the vestiges of original nests were still apparent within the tumor sheets due to a ‘clefting artifact’ outlining the merging nests (Fig. 3I).

Overt squamous differentiation was identified in 2 cases, visible as pink spots on low power in an otherwise blue tumor. In the parotid (case 3), squamous nests, eddies as well as larger keratinized squamous pearls were identified centered abruptly within the monotonous round cells (Fig. 3A–D). Many of the keratinous pearls were calcified. In the second tumor (case 4), the squamous nests were less frequent, smaller, and non-keratinized. Peculiar colonization, rather than destructive infiltration, by tumor cells, was noted (case 1, 2). While in the parotid, tumor cells were seen creeping along and replacing the salivary ducts and the acini (keeping their architecture intact (Fig. 3F–G), the colonizing pattern of spread in the thyroid was more striking. Solid nests of tumor cells were admixed with intact thyroid follicles displaying variably thick collars of neoplastic cells replacing normal follicular cells in a clinging pattern while retaining luminal (colloid) secretions (Figs. 1D, 2F, 3E). Notably, similar follicle-like spaces filled with pale eosinophilic fluid were identified punctuating tumor nests and sheets in two non-thyroidal tumors (Fig. 2D–E). Entrapped ducts were noted in 1 case. Interlobular tumor stroma was hyalinized stroma in the majority while in others it was cellular fibrotic, or loose edematous, or partially myxoid. Mitotic activity and small foci of necrosis were frequent. The overlying mucosa was ulcerated by the tumor in 1 case. No intramucosal pagetoid spread was identified in any case. The underlying bone and cartilage involvement was present in 3 cases. Nodal metastasis was not observed.

Table 3 and Fig. 4 highlight the IHC characteristics of ALES cases. Briefly, the tumors exhibited a consistent profile of diffuse cytokeratin, CD99 (membranous), p40/p63, NKX2.2, FLI-1, and focal synaptophysin positivity. Other markers, performed by the reporting pathologist to exclude a variety of differential diagnoses, including chromogranin, SMARCB1, p16, NUT, LCA, desmin, S100, PAX8, TTF1, WT1, BCOR, calretinin, and calcitonin were negative. Ki-67 ranged from 12–35%. All tumors were positive for EWSR1 rearrangements on FISH (Fig. 4E, F).

Table 3.

Ancillary testing in Adamantinoma-like Ewing Sarcoma cases (n = 7)

Antibody	Case 1	Case 2	Case 3	Case 4	Case 5	Case 5	Case 6	Case 7
AE1/AE3	+	+	+	+	+	+	+	+
EMA	ND	+	ND	ND	ND	ND	+	ND
p40	+	+	+	+	+	+	+	+
CD99 (mic2) membranous	+	+	+	+	+	+	+	+
NKX2.2	+	+	+	+	+	+	+	+
Fli1	+	+	ND	+ weak	ND	ND	+ weak	+
Desmin	-	-	-	-	-	-	-	-
WT1	ND	ND	ND	-	-	ND	-	-
Synaptophysin	+	+ focal	-	-	+	-	+ focal	+ weak
Chromogranin	-	-	-	-	-	-	-	-
NUT	-	-	-	-	-	-	-	-
BCOR	-	ND	ND	ND	ND	ND	ND	-
TTF1	-	ND	ND	ND	ND	ND	ND	ND
PAX8	-	ND	ND	ND	ND	ND	ND	ND
Calcitonin	-	ND	ND	ND	ND	ND	ND	ND
Beta-catenin	ND	-	-	ND	ND	-	-	-
p16 diffuse (> 70%)	-	-	-	-	-	-	-	-
S100/ SOX10	-	-	-	-	-	-	-	-
SMARCB1	ND	ND	-	-	-	-	-	-
Ki67 labeling index	12%	28%	30%	35%	15%	30%	25%	18%
EWSR1 break-apart FISH*	+	+	+	+	+	+	+	+

f focal positivity (< 50% immunoreactivity), ND not done, FISH fluorescent in situ hybridization

^*Ewing sarcoma break point region 1(EWSR1) (22q12.1)

Adjuvant Treatment and Follow Up

Radiotherapy was administered to 4 patients (three adjuvant; one palliative). All patients received chemotherapy [EFT 2001 protocol; VAC/IE (vincristine + doxorubicin + cyclophosphamide and ifosfamide + etoposide) however one patient was lost to follow-up mid-treatment and did not return (case 3). The median follow-up was 26 months (range, 2–39 months; mean, 24.6 months). The response to therapy varied among the five patients who underwent treatment. One patient (case 2) remained disease-free after treatment at 26 months, while another (case 1) developed metastasis after a 36-month disease-free interval. The tumor showed excellent response to chemotherapy in case 6, however, the patient developed progressive disease after 24 months. Two patients developed local recurrences: one patient (case 4) abandoned treatment midway and returned with local recurrence, while the other (case 5) experienced an early recurrence. The time-to-recurrence was 10 and 6 months, respectively. Distant metastases were identified in three patients; the sites of metastasis included brain (in case 1), lung, and skeletal (in case 4), and pancreatic and skeletal (in case 6); the time-to-metastasis (measured from the end of curative treatment to the documentation of metastasis) was 25 months, 6 months, and 0 months, respectively. At the last follow-up visit, two patients were alive with no evidence of disease, three patients were alive with disease, while treatment was ongoing in one patient.

Discussion

ALES is a rare variant of ES that is characterized by EWSR1::FLI1 gene fusions and complex epithelial differentiation; the latter is defined by diffuse and intense immunoreactivity with multiple (high and low molecular weight) cytokeratins and both IHC and ultrastructural evidence of complex desmosomes and tonofilaments [2, 16]. Although ALES exhibits a proclivity to head and neck among other sites, accounting for about 74% of the cases, it is still a rare diagnosis with only 28 cases reported to date [3, 9, 14, 15, 18], summarized in Table 4. The sites previously reported include the major salivary glands (n = 12) [7–9], thyroid (n = 8) [4, 10–15], sinonasal tract (n = 4) [4–6], neck soft tissues (n = 3) [16–18], and orbit (n = 1) [4]. The previously reported cases include one case of head and neck ALES from our institute as a part of 34 cases of Ewing sarcoma with epithelial differentiation; the tumor arose in the neck soft tissues [18]. In the present study, we contribute seven additional cases of molecularly confirmed head and neck ALES (4 in sinonasal, 2 in parotid, and 1 in thyroid) and expand on the existing information on this rare malignancy.

Table 4.

Site-wise literature on Adamantinoma-like Ewing Sarcoma

Site	Author, year	Age/Sex	Initial diagnosis	Treatment S + XRT + CT	Locoregional Recurrence	Metastasis	Outcome	Follow up (months)
Sinonasal region
Nasal/ethmoid	Bishop et al. 2015 [4]	37/F	PD SCC	Initially S; Recurrences: S + XRT + CT (docetaxel, carboplatin, capecitabine, methotrexate)	Yes; 24 months	Yes; Dural, 46 months	DWD	52
Ethmoid sinus, orbit, brain	Bishop 2015 [4]	21/M	ALES	XRT + CT (VDC/IE)	Stable local disease	No	AWD	12
Nasal	Mahadevan 2019 [5]	18/M	Basaloid SCa	NA	NA	NA	NA	NA
Nasal, maxilla, orbit	Alexiev 2017 [6]	41/M	NUT Ca	S + CT (VDC/IE); RT pending	NA	NA	NA	2
Nasal	Bal 2021 [Present series]	30/M	Basaloid SCC	S + XRT; CT at recurrence	Yes	Yes	AWD	39
Maxilla	Bal 2021 [Present series]	27/M	Basaloid SCC	S + XRT + CT	Yes	No	AWD	13
Maxilla	Bal 2021 [Present series]	25/F	Round cell tumor/solid pseudopapillary tumor	CT; Palliative RT	No	Pancreatic and skeletal metastasis (0 months)	AWD	24
Ethmoid/maxilla	Bal 2021 [Present series]	34/F	SNEC	S; Additional therapy pending	NA	NA	NA	0
Salivary
Parotid	Lecanzo [8], Bishop 2015 [4] Rooper 2020 [7]	56/F	Basal cell adenoca	S + XRT + CT (VDC/IE)	No	No	ANED	1
Parotid	Bishop 2015 [4]; Rooper 2020 [7]	40/F	Basal cell adenoma	S; Additional therapy pending	NA	NA	NA	0
Parotid	Rooper 2020 [7]	63/F	PD CA with basaloid features	S + CT (VDC/IE)	No residual	No	DWD	3
Parotid	Rooper 2020 [7]	32/F	High grade neuroendocrine CA	S + XRT + CT (carboplatin/etoposide)	Persistent disease following initial therapy	No	AWD	8
Parotid	Rooper 2020 [7]	32/M	PD CA with basaloid features	S + XRT + CT (VDC/IE)	No residual	No	ANED	19
Parotid	Rooper 2020 [7]	41/M	PD CA with basaloid features	S + XRT + CT (initially carboplatin/paclitaxel then VDC/IE)	No residual	No	ANED	24
Parotid	Rooper 2020 [7]	46/M	Merkel Cell Ca	S; additional treatment pending	No residual	No	NA	0
Parotid	Rooper 2020 [7]	72/M	ALES	S; additional treatment pending	No residual	No	ANED	1
Parotid	Alnuaim 2020 [9]	29/M	-	S + XRT + CT	Yes; 22 months	Pancreatic and skeletal metastasis (22 months)	AWD	22
Parotid	Alnuaim 2020 [9]	46/F	PD NEC	S; additional therapy pending	NA	NA	NA	3
Submandibular	Rooper 2020 [7]	77/M	PD CA with basaloid features	S + XRT + CT (doxorubicin)	No residual	No	ANED	13
Submandibular	Rooper 2020 [7]	58/M	PD Ca	S; additional treatment pending	No residual	No	NA	0
Parotid	Bal 2021 [Present series]	42/M	Basal cell adenoca	S	NA	NA	NA	0
Parotid	Bal 2021 [Present series]	12/F	Sialoblastoma/ES	S + CT	NA	NA	ANED	26
Thyroid
Thyroid	Cruz [10]; Eloy [11]	42/F	Small cell Ca	TT	NO	No	ANED	38
Thyroid	Eloy [11]	24/M	PDTC	TT + XRT + CT	No	No	ANED	156
Thyroid	Bishop 2015 [4]	19/M	ALES	S; additional therapy pending	NA	NA	NA	NA
Thyroid	Bishop 2015 [4]	36/F	ALES	S; additional therapy pending	NA	NA	NA	NA
Thyroid	Ongkeko et al. [12]	36/F	PDTC	S + XRT; CT at metastasis	No	Pancreatic 2 months	ANED	24
Thyroid	Morlete [13]	20/F	ALES	TT + XRT + VDC	No	No	ANED	7
Thyroid	Jones 2020 [14]	21/M	ALES	NA	NA	Multiple lytic bone lesions (? skeletal metastases)	NA	0
Thyroid	Taccogna 2021 [15]	40/M	Undifferentiated thyroid carcinoma	TT	No	No	ANED	9
Thyroid	Bal 2021 [Present series]	7/F	ES	HT + CT + RT	No	Brain, at 25 months	AWD	32
Orbit
Orbit	Bishop 2015 [4]	7/F	Myoepithelial Ca	S + XRT + CT (ifosfamide + cyclophosphamide + etoposide, then ifosfamide + vincristine + etoposide)	No	No	ANED	61
Neck soft tissues
Neck ST and vagus	Weinreb [16]	29/M	PDSCC	66 Gy RT + 3# cisplatin. Later switched to VAC/IE	No	No	ANED	-
Vagus	Kikutchi [17]	11/F	Malignant tumor	S + CT; S + RT (at recurrence)	Y	No	AWD	36
Neck soft tissues	Bharat Rekhi [18]	36/M	-	S + XRT + CT	No	No	AWD	17

M male, F female, PDSCC poorly differentiated squamous cell carcinoma, S surgery, XRT external beam radiotherapy, Rt radiotherapy, CT chemotherapy, ALES Adamantinoma-like Ewing Sarcoma, Ca carcinoma, BSCC basaloid squamous cell carcinoma, SNEC sinonasal neuroendocrine carcinoma, ES Ewing sarcoma, PD Ca poorly differentiated carcinoma, TT total thyroidectomy, HT hemithyroidectomy, VDC/IE alternating vincristine/doxorubicin/cyclophosphamide and ifosfamide/etoposide, NA not available, DWD died with disease, AWD alive with disease, ANED alive with no evidence of disease, ST soft tissues;

While the age spectrum of ALES is wide (range 7–77 years), most (> 80%) patients are younger than 50 years of age (mean, 36.7 years; median, 36 years) [3–18]. Patients in our cohort were comparatively young, with an average age of 25.2 years (all were < 50 years of age). Site-wise age variations have been noted. The average age of salivary ALES patients has been observed to be greater than for non-salivary sites (49.3 years versus 25.8 years, respectively) [3–18]. Such site-specific age differences were, however, not appreciated in our small series. A minor male preponderance has been previously recorded (M: F, 1.2) [3–18], similar to our findings. Symptoms may be non-specific; most patients present with an enlarging mass, with or without pain, while sinonasal tumors mostly cause obstruction, pain, and/ or epistaxis. Rare instances of cervical sympathetic chain involvement leading to Horner syndrome and vocal cord palsy have been reported [16]. Exceptionally, metastatic disease may be the first presenting site. One of our patients presented with recurrent abdominal pain and was diagnosed as a solid pseudopapillary tumor in a pancreatic biopsy elsewhere. However, complete workup revealed a large primary in the maxilla and multiple skeletal and pancreatic metastases.

ALES may present with a variable tumor size that ranges from 2.2–7.9 cm [3, 4, 7, 9]. Macroscopic appearance usually reveals a grey-white tumor with a firm, lobulated cut surface and variable necrosis, calcification, and cystic change [3]. While most tumors may be organ confined, their borders tend to be infiltrative [3]. Histologic features of ALES are quite distinctive albeit encompassing a wide morphologic spectrum. Similar to ES, ALES is characterized by a monotonous population of round cells and CD99 (mic2) and NKX2.2 positivity. However, unlike typical ES, a nested growth pattern, peripheral palisading, and a complex epithelial differentiation (evinced by low- and high-molecular-weight cytokeratins, and p40/p63 immunoreactivity, with or without overt squamous differentiation, or keratin pearls) bestow ALES a deceptive resemblance to carcinomas [3, 4, 7]. These hybrid pathologic features of ES and carcinoma make ALES a distinctive and treacherous entity at the same time. Akin to most translocation-associated tumors, the neoplastic cells are characteristically monotonous and possess isomorphic nuclei, inconspicuous nucleoli, and scanty pale to basophilic, or clear cytoplasm [4, 7, 16]. Pseudo-rosettes are identified in more than half the cases, although their presence is usually focal [3, 4]. Tumors show brisk mitoses while necrosis is variable, ranging from sparse to extensive and geographic [16].

One of the distinctive features of diagnostic importance, identified in about a third of ALES cases [4–7, 9, 16, 17] is the presence of overt squamous differentiation. The latter may be present as rare isolated keratinized cells, or as variable-sized squamous nests, eddies, and sometimes as keratin pearls. Irrespective of their frequency, these squamous foci are conspicuous for their abrupt apposition to the monotonous round cells [4, 5, 7]. This appears to be least frequent at major salivary locations as about two-thirds of ALES cases with squamous differentiation have been reported at non-salivary sites [7]. Of our two cases with morphologic evidence of squamous differentiation, one was in the nasal cavity and the other in the parotid. Mixed architectural patterns comprising nests, lobules, trabeculae, and sheets are usually present with the nested pattern, even if focal, being consistent [4, 7]. Typically, these nests are separated by fibrous stroma which is mostly hyalinized and rarely may display a microcystic myxoid appearance [4]. Further, a subset of cases may display a hyaline basement membrane-like material which can erroneously sway the diagnosis towards a salivary gland tumor [4, 7]. Myxoid stroma or basement membrane-like material was not identified in any of our cases. Uncommonly, stromal osteo-fibrous dysplasia-like metaplasia [6], chondroid metaplasia [9], pseuodoglandular pattern [17], vague streaming pattern [4], and a biphasic pattern with a spindle component have been described [17].

Another intriguing feature described by many authors is the peculiar colonization pattern of spread in ALES, documented in approximately 25% of cases [4, 6, 9, 13]. Rather than a destructive infiltration into adjoining tissues, tumor cells display a clinging/ pagetoid pattern of spread along the pre-existing structures such as adjacent thyroid follicles, salivary ducts, and acini. This pattern has been most consistently reported in the thyroid where it may serve as a strong diagnostic clue (Figs. 1D, 2F, 3E.) [4, 13]. Similarly, an intraepithelial and intraductal pagetoid pattern of tumor spread, analogous to carcinomas, has been reported in the sinonasal mucosa [4, 6] and salivary ducts [9]. Further, some authors have described a permeative tumor cell infiltration with entrapment of normal ducts and thyroid follicles [11, 12]. We identified a similar colonization pattern of spread in two of our cases (Fig. 3F–G). Interestingly, follicle-like spaces formed by small cysts lined by neoplastic cells and filled with pale eosinophilic fluid were seen punctuating solid sheets in two of our non-thyroidal cases (Fig. 2D, E). Also, in one tumor a peculiar clefting artifact was seen rimming the coalescent nodules (Fig. 3I). Follicle-like spaces and clefting artifacts have not been previously reported in the literature and their significance as a specific finding remains to be seen. Lymphovascular invasion (LVI) has been identified in a few cases (~ 12%) [9, 10, 12]. Also, nerve infiltration has been observed in about one-third of cases [3, 4, 16, 17]; there are reports of facial nerve involvement in a parotid tumor [9] and vagus nerve infiltration in the neck tumors [16, 17]. We did not encounter nerve involvement or LVI in our cases. Two of our cases had R1 (microscopic margin positivity) resections. The impact of margins in ALES has not been evaluated adequately, although a complete resection seems logical. Lymph node involvement is remarkably low with most instances being reported at recurrences [12, 17]. None of our cases had regional lymph nodal involvement at presentation.

Rarity of occurrence leading to a lack of familiarity, exacerbated by overlap with diverse, and more frequently occurring pathologic entities make ALES particularly susceptible to misdiagnosis in the head and neck. The initial diagnosis in 20 out of 26 cases (where information is available) reported in the literature and 6 cases in the present series were erroneous (Table 4). On one hand, squamous differentiation, keratin pearls, basaloid cells, and peripheral palisading, renders ALES remarkably similar to carcinomas, especially basaloid squamous carcinoma or a poorly differentiated squamous cell carcinoma (SCC). Conversely, small round cell morphology of ALES can bring a plethora of small round cell tumors into the list of differential diagnoses, such as Ewing sarcoma (including CIC-rearranged sarcomas, BCOR-sarcomas, and round cell sarcomas with EWSR1-non-ETS fusions), lymphoma/leukemia, alveolar rhabdomyosarcoma, synovial sarcoma (poorly differentiated), melanoma, Merkel cell carcinoma, neuroendocrine carcinomas (NEC), and desmoplastic small round cell tumor (DSRCT), etc. [3, 4]. Furthermore, the tumor location brings additional site-specific differential diagnoses into consideration. In the salivary gland (due to basaloid cells, basement membrane-like material, and palisading), ALES may mimic primary salivary neoplasms such as basal cell adenocarcinomas/adenomas (BCAC/BCA), myoepithelial neoplasms, solid adenoid cystic carcinoma, sialoblastoma, SCC, and NEC [3, 4]. Similarly, sinonasal ALES may be mistaken for basaloid/ poorly differentiated SCC, and NUT carcinoma (due to abrupt squamous differentiation/ keratinization) sinonasal NEC, sinonasal undifferentiated carcinoma, olfactory neuroblastoma, SMARCB1/SMARCA4-deficient carcinoma, HPV-multiphenotypic sinonasal carcinoma, or rarely, teratocarcinosarcoma (due to neuroectodermal and squamous elements in a biopsy). In the thyroid, poorly differentiated thyroid carcinoma, medullary thyroid carcinoma, and carcinoma with thymus-like elements (CASTLE) are the differential diagnoses. Periorbital soft tissue ALES can be mistaken for a sebaceous carcinoma, basal cell carcinoma, or basosquamous carcinoma. Rarely, hybrid features of neuroectodermal/endocrine and epithelial differentiation may be misdiagnosed as mixed neuroendocrine non-neuroendocrine neoplasm (MiNEN). Using appropriate ancillary tests can help in distinguishing ALES from its morphologic mimics. Table 5 highlights the IHC profiles and clinically relevant molecular alterations of the diverse histologic mimics of ALES at various sites.

Table 5.

Ancillary testing in differential diagnoses of Adamantinoma-like Ewing Sarcoma

Tumor	Immunophenotype	Clinically relevant Molecular alterations	References
Adamantinoma-like Ewing Sarcoma	CD99, NKX2.2, AE1/AE3, p40, p63, HMWCK, Fli1 (v), Synaptophysin (v), Chromogranin (v), p16 (v)	EWSR1::FLI1	[2–18, 22]
Predominantly round cell morphology [2, 19–30]
Classic Ewing sarcoma	CD99 (membranous), NKX2.2, Fli1 (fusion pos), ERG (fusion pos), AE1/AE3 (25%), S100 (-/ +)	EWSR1::FLI1 ((about 85%), EWSR1::ERG (about 10%), others members of FET (TAF15, FUS, EWSR1) and ETS family	[2, 19–22]
Sarcoma with BCOR	BCOR, SATB2, TLE1, cyclin D1, CD99 (50%)	BCOR rearrangements (mostly, BCOR::CCNB3); BECOR-ITD	[22, 23]
CIC-related sarcoma	WT1, CD99 (focal; 20% diffuse), ETV-4, calretinin (v), ERG (v) Occ pos-Keratins, S100, desmin; CIC-NUTM1 express NUT NKX2.2 is negative	CIC::DUX4 (95%); CIC::NUTM1	[22, 24, 25]
Round cell sarcoma with EWSR1-non-ETS	Variable co-expression of myogenic markers (desmin, myogenin, MYOD1) and neurogenic markers (S100P, SOX10, MITF, GFAP) CD34 can be positive CD99 is not consistently expressed	EWSR1::NFATC2, FUS::NFATC2, and EWSR1::PATZ1	[22, 26, 27]
Alveolar Rhabdomyosarcoma	Desmin, Myogenin, MyoD1, CD99 (cytoplasmic ±), Occ pos- AE1/AE3, synaptophysin, chromogranin, CD56 (pitfall)	85% fusion; PAX3::FOXO1 (70–90%); PAX7::FOXO1 (10–30%)	[22, 28]
Desmoplastic small round cell tumor	AE1/AE3, desmin, WT1 (C-terminus antibody), NSE, CD56, CD99 (v) Myogenin and MYOD1 are consistently negative	EWSR1 gene rearrangement; EWSR1::WT1 fusion	[22, 29]
Non-Hodgkin Lymphoma/ Leukemia	LCA, B cell or T cell lineage markers, Tdt (leukemic blasts)	Lymphoma specific gene fusions	[22]
Poorly differentiated Synovial sarcoma	EMA, keratins, SS18-SSX fusion specific antibody, TLE1, bcl-2, CD99 (v), S100 (v, focal)	SS18::SSX1/2/4	[22, 30]
Predominantly neuroendocrine/ neuroectodermal
Olfactory neuroblastoma	Synaptophysin, chromogranin, SSTR2, calretinin, AE1/AE3 (-/ + , 1/3rd focal), S100 positive rim Negative- CD99	–	[31]
Sinonasal neuroendocrine carcinoma	AE1/AE3, Synaptophysin, chromogranin		[3, 4]
Teratocarcinosarcoma (in a biopsy; sampling of the neuroectodermal components)	Neuroectodermal component-CD99 (cytoplasmic), synaptophysin, INSM1, Chromogranin (v), GFAP (v) Squamous- p40/p63 +  Epithelial- AE1/AE3, Ck7 (v) Mesenchymal- Vimentin, desmin (v), MyoD1 (v)	SMARCA4 deletions (subset)	[32]
Melanoma	HMB45, S100, SOX10, Melan A, TIFF	KIT, RAS, BRAF mutations	[33]
Merkel cell carcinoma	Cytokeratins, synaptophysin, chromogranin, NSE, INSM1, CD56, NFP, CK20 (perinuclear dot), CM2B4 (anti-MCPyV)	MCPyV DNA	[3, 4, 34]
Predominantly carcinoma morphology
Basaloid squamous carcinoma	Cytokeratins, p40, p63, HMWCK	–	[3, 4]
NUT carcinoma	NUT, AE1/AE3, p63 > p40, HMWCK, synaptophysin and, chromogranin (occasional), CD34 (v)	NUT::BRD4; NUT::BRD3; NUT::NSD3;	[35]
SMARCB1-deficient carcinoma	SMARCB1 loss, AE1/AE3, p40, p63, HMWCK, CK7 (v) Synaptophysin and chromogranin (v, 8–18%)	Biallelic SMARCB1 deletions (2/3rds)	[4, 36]
SMARCA4-deficient carcinoma	SMARCA4 loss, AE1/AE3, CK7 (rare), synaptophysin (90%), chromogranin (40%), CD56 (60%)	Biallelic SMARCA4 inactivation	[4, 36]
Sinonasal undifferentiated carcinoma	AE1/AE3, CK7, p63 (v), p16 (v), IDH1/2 mutant specific (subset)	IDH2-mutations (33–85%)	[3, 4, 37]
Basal cell adenocarcinoma	Cytokeratins, p40, p63, S100, SOX10, beta-catenin	CYLD or CTNNB1 alterations (subset)	[7, 38]
Myoepithelial carcinoma	Cytokeratins, p40, p63, S100, SOX10, SMA, calponin	EWSR1::POU5F1, PBX1, PBX3 or ZNF444 Clear cell MECA- EWSR1 rearrangements	[7, 39]
Sialoblastoma	Cytokeratins (v), SOX10, p63, beta-catenin, S100 (v), SMA (v), CD117 (v), and calponin (v)	–	[40]
Solid Adenoid cystic carcinoma	Cytokeratins, CK7, SOX10, S100, SMA, p40, p63, Calponin	MYB/MYBL1 gene rearrangements or fusions	[4, 7, 41]
Ameloblastic Carcinoma	Cytokeratins, p40, p63, CK19, calretinin, SOX2 (v), SMA (v), abnormal p53	BRAF V600E; TP53	[42]
Sebaceous carcinoma	EMA, p40, p63, p16, AR, adipophylin, p53, perilipin	TP53/RB1 mutations; HER2 amplification (75%)	[43, 44]
Basal cell carcinoma/basosquamous carcinoma	BerEP4, p40, p63, bcl-2, synaptophysin (v), chromogranin (v)	–	[45]
Mixed neuroendocrine non-neuroendocrine neoplasm (MiNEN)	Synaptophysin, chromogranin, INSM1 in the NE component; Cytokeratins in non-NE, p40 if squamous component	–	[46]
Medullary thyroid Carcinoma	Calcitonin, INSM1, Synaptophysin, Chromogranin, CEA, TTF1 (v)	RET gene rearrangements	[47]
Poorly differentiated thyroid carcinoma	AE1/AE3, TTF1, PAX8, Thyroglobulin positive	–	[3, 4]
CASTLE	CD5, p63, CD117, synaptophysin (v), chromogranin (v)	–	[3, 48]

v variable positivity, pos positive, ITD Internal tandem duplication, occ occasional, NE neuroendocrine, CASTLE Carcinoma with thymus-like elements;

ALES exhibits a characteristic immunoprofile of strong/diffuse, membranous CD99 (mic2) and nuclear NKX2.2 positivity combined with diffuse and strong cytokeratin (including high-molecular-weight), p40, and p63 positivity [2–4]. Fli-1 and synaptophysin positivity (usually focal) may be seen in up to half the cases; the latter has been observed more frequently in salivary ALES (observed in 81% cases) [7]. While classic ES may demonstrate cytokeratin (mostly low molecular-weight) positivity in up to 25–30% cases [19–21], diffuse p40, p63, and HMWCK reactivity are not seen. Interestingly, variable p16 positivity, including diffuse [9], has been reported in ALES, however when investigated, no association with HPV has been discovered [4, 11]. ALES is consistently negative for NUT, desmin, LCA, SOX10, Melan A, SMA, WT1, BCOR, beta-catenin (nuclear), TLE1, PAX8, and TTF1 [2–4]. Ki67 labeling index ranges from 12–35% [6, 9]. p53 expression is usually wild type [16].

ALES share their molecular profile with classic ES that is defined by FET::ETS gene rearrangements [2]; the FET family of transcription factors include EWSR1 or FUS and TAF15 while the ETS family comprises FLI1, ERG, ETV1, E1A-F, and FEV [2]. All cases of ALES have been reported to harbor the t(11;22) translocation with EWSR1::FLI1 fusion by either FISH, reverse transcriptase-polymerase chain reaction (RT PCR), or next-generation sequencing [2, 3]. While this translocation or EWSR1 rearrangement on break-apart FISH is diagnostic, it is noteworthy that many other distinct tumor entities, such as, myoepithelial carcinomas, hyalinizing clear cell carcinomas, EWSR1-non-ETS sarcomas, and DSRCT may also harbor EWSR1 rearrangement [3]. Conversely, it is plausible that genes other than EWSR1, such as FUS, or TAF-15 belonging to the FET family may be involved, although this has not been reported so far for ALES. Two of our cases (not included in the present study) with classical histopathologic and IHC features of ALES were negative for EWSR1 break-apart FISH. However, a lack of further investigation precluded the determination of the underlying genetic alteration.

Knowledge of the clinical course of ALES and the optimal treatment is limited as few studies are available with long-term follow-up. Most patients of ALES have received multi-modality treatment comprising surgery, RT, and CT; latter usually on the lines of ES treatment using VAC or VDC alternating with IE [3]. Out of 26 previously published cases, treatment and outcome information is available in only 18 patients with a median follow-up duration of 13 months (mean 26.7 months; range 7–156 months) [3–18]. Of these 18 patients, local recurrences/persistent disease was noted in 27.8% of patients while 17% of patients developed distant metastasis [3–18]. The sites of metastasis reported were dura [4], pancreas, and skeletal [9, 12]. Two-thirds were alive with no evidence of disease, 22.2% were alive with the disease while 11.1% had died of the disease [3–18]. Among our patients, the recurrence rate was 33.3% while three (50%) of our patients developed disseminated disease with sites of metastases being brain, pancreas, skeleton, and lung. In our study with a median follow-up of 26 months, 40% of patients were alive with no evidence of disease while 60% were alive with the disease. In our small cohort of patients treated with multimodality treatment, the high rate of disease relapse suggests aggressive biology. Studies on larger patient cohorts are warranted to decipher the long-term outcomes of this rare malignancy.

Our study had limitations of a retrospective study, a small sample size, and inadequate follow-up. Nonetheless, the present study adds seven additional cases of a rare malignancy that further expands the knowledge of the morphologic and clinical spectrum of ALES while underscoring the need to avoid diagnostic errors. To the best of our knowledge, this is the largest study from the South-Asian region in the Indian population. We also report for the first time the presence of follicle-like spaces in these tumors that may serve as a useful diagnostic clue.

Conclusion

ALES is a rare malignancy with distinctive pathologic features and a strong predilection for the head and neck region. Nested architecture, cellular monotony, complex epithelial differentiation, and EWSR1::FLI1 translocation are the pathologic hallmarks of ALES. The peculiar tendency towards the colonization of native structures and the presence of follicle-like spaces may serve as useful diagnostic clues. Further, the awareness that abrupt squamous differentiation with keratinization is a feature seen in a subset of ALES may avert diagnostic pitfalls when interpreting head and neck tumor biopsies. Whether ALES belongs with ES or is an independent entity sharing the same translocation is debatable. Nonetheless, ALES displays a unique set of pathologic features that make it a distinctive clinicopathologic entity. Its biology and optimal treatment protocols need to be defined and warrant larger prospective multicentric studies.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

The study conception and design were by Munita Bal. Provision of study materials or patients: Munita Bal, Aekta Shah, Neha Mittal, Katha Rabade, Swapnil Rane, Gauri Pantavaidya, Deepa Nair, Sarbani Ghosh Laskar, Siddharth Laskar, Aishwarya Malla, Krishna Kumar, Prabhash Kumar. Collection and assembly of data: Munita Bal, Aekta Shah. Data analysis and interpretation: Munita Bal, Asawari Patil, Aekta Shah, Bharat Rekhi, Omshree Shetty. The first draft of the manuscript was written by Munita Bal and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

No funding obtained.

Declarations

Conflict of interest

No conflict of interest to disclose.

Ethical Approval

All procedures performed in the study were in accordance with the ethical standards of the Institutional Ethics Committee (IEC). A waiver of review was granted by the IEC after due examination.

Informed Consent

Not required as per institutional ethics committee’s policy for retrospective case series. The authors declare that all information is anonymized and the submission does not include images that may identify any patient.