cancers

Article
Neuroendocrine Carcinoma of the Larynx and Pharynx:
A Clinical and Histopathological Study
Primož Strojan 1, *,† , Robert Šifrer 2 , Alfio Ferlito 3,† , Cvetka Grašič-Kuhar 4 , Boštjan Lanišnik 5 ,
Gaber Plavc 1 and Nina Zidar 6,†

1 Department of Radiation Oncology, Institute of Oncology Ljubljana and Faculty of Medicine,

University of Ljubljana, 1000 Ljubljana, Slovenia; gplavc@onko-i.si
2 Department of Otorhinolaryngology and Cervicofacial Surgery, University Medical Centre Ljubljana and
Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia; robert_sifrer@hotmail.com
3 Coordinator of the International Head and Neck Scientific Group, 35100 Padua, Italy;
profalfioferlito@gmail.com
4 Department of Medical Oncology, Institute of Oncology Ljubljana and Faculty of Medicine,
University of Ljubljana, 1000 Ljubljana, Slovenia; cgrasic@onko-i.si
5 Department of Otolaryngology, Head and Neck Surgery, University Medical Center Maribor,
2000 Maribor, Slovenia; bostjan.lanisnik@ukc-mb.si
6 Institute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia;
nina.zidar@mf.uni-lj.si
* Correspondence: pstrojan@onko-i.si; Tel.: +386-1-5879290
† This article was written by members and invitees of the International Head and Neck Scientific Group
(www.IHNSG.com).



Simple Summary: Neuroendocrine carcinomas (NECs) of the head and neck are rare. The presented


series of 20 patients with laryngeal and pharyngeal NECs is population-based and one of the
Citation: Strojan, P.; Šifrer, R.; Ferlito, largest published to date. We analyzed the treatment results according to the type of therapy
A.; Grašič-Kuhar, C.; Lanišnik, B.; and the role of various standard (synaptophysin-chromogranin-CD56, Ki-67, p16, HPV, and EBV)
Plavc, G.; Zidar, N. Neuroendocrine
and some novel (INSM1 and PD-L1) neuroendocrine markers or potential prognosticators. The
Carcinoma of the Larynx and
results indicate the following: (1) laryngeal and pharyngeal NECs accounted for 0.43% and 0.17%
Pharynx: A Clinical and
of the cases in the corresponding tumor groups, respectively; (2) neuroendocrine differentiation
Histopathological Study. Cancers
can be reliably determined by INSM1 immunohistochemistry; (3) the prognosis was determined
2021, 13, 4813. https://doi.org/
10.3390/cancers13194813
by the nodal stage and TNM stage but not by the histological grade (which refers to moderately
and poorly differentiated NECs); (4) except in well-differentiated NECs and early-stage (T1-2N0-1)
Academic Editor: Gino Marioni moderately/poorly differentiated NECs, aggressive multimodal therapy is needed; and (5) the p16,
HPV, and EBV statuses failed to show any prognostic value.
Received: 9 August 2021
Accepted: 23 September 2021 Abstract: Neuroendocrine carcinomas (NECs) of the head and neck are rare and the experience
Published: 27 September 2021 scanty. The Cancer Registry of Slovenia database was used to identify cases of laryngeal and
pharyngeal NECs diagnosed between 1995–2020. Biopsies were analyzed for the expression of
Publisher’s Note: MDPI stays neutral standard neuroendocrine markers (synaptophysin, chromogranin, CD56), INSM1, Ki-67, p16, and
with regard to jurisdictional claims in PD-L1 (using the combined positive score, CPS). In situ hybridization for human papillomavirus
published maps and institutional affil-
(HPV) and Epstein–Barr virus (EBV) was performed. Twenty patients (larynx, 12; pharynx, 8) were
iations.
identified. One tumor was well differentiated (WD), five were moderately differentiated (MD), and
14 were poorly differentiated (PD). Disease control was achieved solely by surgery in 4/4 MD/PD
T1-2N0-1 tumors. Eight patients died of the disease, seven of which were due to distant metastases.
All three traditional markers were positive in 11/17 NECs and the INSM1 marker in all 20 tumors.
Copyright: © 2021 by the authors.
Two of fourteen p16-positive tumors were HPV-positive, but all three nasopharyngeal NECs were
Licensee MDPI, Basel, Switzerland.
EBV-negative. Three tumors had CPSs ≥ 1. In conclusion, INSM1 was confirmed to be a reliable
This article is an open access article
marker of neuroendocrine differentiation. Except in WD and early-stage MD/PD tumors, aggressive
distributed under the terms and
conditions of the Creative Commons
multimodal therapy is needed; the optimal systemic therapy remains to be determined. p16, HPV,
Attribution (CC BY) license (https:// and EBV seem to bear no prognostic information.
creativecommons.org/licenses/by/
4.0/).

Cancers 2021, 13, 4813. https://doi.org/10.3390/cancers13194813 https://www.mdpi.com/journal/cancers

Cancers 2021, 13, 4813 2 of 16

Keywords: neuroendocrine carcinoma; head and neck cancer; diagnosis; therapy; prognosis

1. Introduction
Primary neuroendocrine carcinomas (NECs) of the head and neck (HN-NEC) are rare.
Their exact incidence is not known, and only estimates are available for the most common
subgroups of these tumors, such as small-cell NECs (SCNECs) or those growing in the
larynx, representing 0.27% and 0.38% of the head and neck tumor cases [1,2]. In addition,
changes in the HN-NEC nomenclature over time have further contributed to the ambiguity
regarding these tumors: they have mainly affected the group of moderately differentiated
(MD) HN-NECs, putting into question the relevance of older literature data [3,4]. For
example, a large-cell NEC (LCNEC) was defined as an independent histological entity in
2005 and was traditionally not separated from other MD HN-NECs [5–8]; only since 2017
has LCNEC been formally classified in the poorly differentiated (PD) category [9].
Due to their sporadic occurrence, only reports of individual cases or small patients’
series covering longer periods are available, which does not allow for a comprehensive
analysis of the diagnostic and/or prognostic significance of individual histological markers
or treatment approaches [10,11]. The diagnosis of HN-NEC is challenging and requires
strict adherence to the 2017 WHO classification criteria, including the use of a panel of
immunohistochemical markers to correctly classify these tumors [9]. However, the utility
of traditional neuroendocrine markers (synaptophysin, chromogranin, and CD56) can
be limited due to their wide range of sensitivities (individual or combined, 50–80%) and
expression in tumor types other than neuroendocrine neoplasms, as well as the uncertain
fidelity of cytoplasmic staining (i.e., weak and/or focal reactivity) [12].
In the HN-NEC patients, the treatment and survival results correlate with the disease
stage, which is primarily determined by the degree of tumor differentiation and has not
significantly changed with advances in diagnostics and treatment (e.g., the introduction of
modern radiotherapy (RT) techniques and systemic treatments). While the disease-specific
survival (DSS) for the notoriously rare well-differentiated (WD) HN-NECs is excellent,
fewer than 20% of patients with PD tumors survive for five years after diagnosis [10,13].
As the curative potential of local therapies (surgery and radiotherapy) is eventually limited
to the very early stage of the disease, in the majority of patients, the addition of systemic
therapy is indicated to counteract microscopic or clinically overt metastases. While most
tumors display an initial response to platinum-based chemotherapy (ChT) regimens, re-
sistance develops at early timepoints during the course of disease, resulting in limited
prognosis [14,15]. Thus, reliable prognostic markers and new therapeutic options are
needed to improve the outcomes.
The present study aimed to review the experience with laryngeal and pharyngeal
NECs that were diagnosed in Slovenia between 1995 and 2020. Special emphasis was placed
on the pattern of treatment failure and survival depending on the treatment received and
the role of standard and certain novel histological markers for the diagnosis and prognosis
of the disease.

2. Materials and Methods

2.1. Patients
The study included patients with diagnosed NEC of the larynx or pharynx in Slovenia
between 1995 and 2020. Cases of NEC were identified from a computerized search of the
Cancer Registry of Slovenia database (https://www.onko-i.si/eng/sectors/epidemiology-
and-cancer-registry, accessed on 9 August 2021), which is the state’s central service for
collecting and managing data on new cancer patients. For each case, paraffin blocks were
referred to the Institute of Pathology at the Faculty of Medicine Ljubljana for a central
review to confirm the diagnosis of NEC and for grading according to the 2017 World Health
Organization Classification of Head and Neck Tumors [9]. A representative block was
Cancers 2021, 13, 4813 3 of 16

chosen for immunohistochemical and in situ hybridization studies. The medical records of
identified patients were reviewed to collect information on the characteristics of patients
and tumors, therapy, and disease outcome. The tumors were staged using the criteria of
the International Union against Cancer (UICC) TNM staging system, 7th edition [16].

2.2. Histological and Molecular Analyses

Biopsy tissue samples were fixed in 10% buffered formalin for 24 h, embedded in
paraffin, cut at 4 µm, stained with hematoxylin and eosin, and analyzed according to
standard criteria. Some immunohistochemical analyses and in situ hybridization was
performed as part of the standard diagnostic procedures, and some were performed during
this study.

2.2.1. Immunohistochemistry
For immunohistochemistry, we used commercially available antibodies presented in
Table 1. Staining was performed in the BenchMark XT immunostainer (Ventana Medical
Systems, Tuscon, AZ, USA). After antigen retrieval with Cell Conditioning 1 buffer (Ventana
Medical Systems) at 95 ◦ C for 30 min, the slides were incubated with primary antibodies
against the antigens. The immunoreactivity was visualized using the iVIEW DAB Detection
Kit (Ventana Medical Systems), according to the manufacturer’s instructions. Sections were
counterstained with hematoxylin. Positive controls and negative controls omitting the
primary antibodies were also included.

Table 1. Overview of source and clone, dilution of the primary antibodies, and antigen retrieval
methods used for immunohistochemistry.

Antigen Clone Manufacturer Pretreatment Dilution

CD56 MRQ-42 Cell Marque, Rocklin, CA, USA CC1 60 min Ready to use
Chromogranin polyclonal Dako, Glostrup, Denmark CC1 60 min 1:1000
Cytokeratin
AE1AE3 Novocastra, Wetzlar, Germany CC1 60 min 1:50
AE1/AE3
Santa Cruz Biotechnology,
INSM1 A-8 CC1 56 min 1:50
Santa Cruz, CA, USA
Ki-67 MIB-1 Dako, Glostrup, Denmark CC1 60 min 1:50
p16 E6H4 Ventana, Tuscon, AR, USA CC1 32 min Ready to use
PD-L1 SP263 Ventana, Tuscon, AR, USA CC1 56 min Ready to use
Synaptophysin MRQ-40 Cell Marque, Rocklin, CA, USA CC1 64 min Ready to use

p16 immunohistochemistry was considered positive if strong and diffuse nuclear and
cytoplasmic expression was found in at least 75% of the tumor [17].
PD-L1 immunohistochemistry was determined by using the combined positive score
(CPS), defined as the number of PD-L1-staining cells (tumor cells, lymphocytes, and
macrophages) divided by the total number of viable tumor cells, multiplied by 100. The
specimen was considered to have PD-L1 expression if CPS ≥ 1 [18].

2.2.2. In Situ Hybridization

The HPV E6/E7 mRNA transcripts were analyzed using the commercially available in
situ hybridization RNAscope 2.0, an HPV HR7 kit (Advanced Cell Diagnostics, Hayward,
CA, USA), and targeting the E6/E7 mRNA of the HPV types 16, -18, -31, -33, -35, -52,
and -58. The reactions were performed following the manufacturer’s instructions. Briefly,
4–5 µm tissue sections were first incubated at 60 ◦ C for 1 h, followed by deparaffinization
and incubation with pretreatment reagent 1 for 10 min at room temperature, pretreatment
reagent 2 for 15 min at 100 ◦ C, and pretreatment reagent 3 for 30 min at 40 ◦ C. The tissue
sections were then incubated with an HPV probe cocktail for 2 h at 40 ◦ C. The hybridization
complexes were visualized using the Detection Kit ACD (Advanced Cell Diagnostics,
Cancers 2021, 13, 4813 4 of 16

Hayward, CA, USA). The sections were counterstained with hematoxylin. A positive
reaction was defined as punctate brown staining in the nuclei and/or cytoplasm [19].
In situ hybridization for the Epstein–Barr virus (EBV) was performed using the
INFORM EBER Probe and the Ventana BenchMark XT Ultra automatic immunostainer
following the manufacturer’s recommendations (Ventana Medical Systems, Tucson, AZ,
USA). EBER-ISH was performed with an ISH iView Blue Detection Kit. Protein removal and
nucleic acid exposures used ISH protease 3 (#780-4149). Counterstaining was performed
with Red Counterstain II. For heat-induced epitope retrieval (HIER), the treatment time
for cell conditioning (CC2) was varied. A positive reaction was defined as blue staining in
the nuclei.

2.3. Statistics
The results were analyzed using the PC SPSS (Release 27, SPSS Inc., Chicago, IL,
USA) statistical package. All of the tests were two-sided, and the results were considered
significant at a probability level of ≤5%.
Basic descriptive statistics are reported as median (range) values for numerical vari-
ables and as percentages for categorical variables. An unpaired Student’s t-test was used
for comparing age between groups, and the Pearson chi-square test was employed for
statistical comparisons for categorical data. In the case of expected parameter values of
<5 in >20% of cells, Fisher’s exact test was used, which facilitates the analysis of smaller
population sizes. The follow-up period and survival were calculated from the date of
diagnosis to 30 April 2021 (close-out date), or an event (relapse or death), whichever came
first. Local tumor control was defined as a complete and permanent eradication of the
tumor in the treatment area. The patients who died of progression of locally persistent
disease after initial therapy were considered to have a local/regional event-free interval of
zero. The Kaplan–Meier product limit method was used for a statistical assessment of local
failure-free survival (LFFS), distant metastasis-free survival (DMFS), NEC-relapse-free sur-
vival (NEC-RFS, locoregional and distant failure considered as an event), disease-specific
survival (DSS, deaths from disease unrelated causes censored), and overall survival (OS,
all deaths considered as events). The differences between the groups were compared using
the log-rank test.

3. Results
3.1. Patients
Over a period of 26 years, 20 patients with NECs of the pharynx or larynx were
identified. The majority were men (15, 75%), former or active smokers (15, 75%), and
aged 26–87 years (median: 64 years). The origin of 12 (60%) tumors was the larynx, three
primary tumors each originated in the nasopharynx and hypopharynx, and two originated
in the oropharynx, which represented 0.43% of all the laryngeal tumors (International
Classification of Diseases, 10th revision (ICD-10) code C32) and 0.17% of all the pharyngeal
tumors (ICD-10 codes C01 and C09–C14) diagnosed in Slovenia during this period [20].
Ten (50%) patients presented with a regional disease, and one (5%) patient presented with
systemic metastases (lung).
Histologically, there were 1 WD NEC, 5 MD NECs (Figure 1), and 14 (70%) PD NECs
(large cell, eight; small cell, six) (Figures 2 and 3). One patient (Case 10) had a mixed
laryngeal primary tumor consisting of LCNEC and squamous cell carcinoma. Patients with
PD NECs more often had advanced disease at presentation (TNM Stage III-IV, WD + MD
vs. PD: 2/6 vs. 12/14, p = 0.04) and were heavier smokers (defined as >15 pack-years
(N = 17), WD + MD vs. PD: 0/6 vs. 8/11, p < 0.01). Early-stage disease (TNM Stage I–II)
was more often diagnosed in females (females vs. males: 4/5 vs. 2/15, p = 0.01) and
in patients with laryngeal primary tumors (laryngeal vs. other primaries: 6/12 vs. 0/8,
p = 0.04). Patients with smaller primary tumors (Stage T1-2) were treated more frequently
with upfront surgery (T1-2 vs. T3-4: 9/10 vs. 3/10, p = 0.02).
Cancers 2021, 13, 4813 5 of 16
Cancers 2021, 13, x 6 of 15
Cancers 2021, 13, x 6 of 15

The detailed data on the characteristics of the patients and tumors, therapy, and
DOC—died of other causes. One Patient received 26 of the planned 28 fractions of postoperative radiotherapy due to the
DOC—died
developmentofofother causes.
bacterial survival
Onewhich
sepsis, are
Patient presented
received
was in the
Table
26cause
also the of 2.
ofplanned
death. 28 fractions of postoperative radiotherapy due to the
development of bacterial sepsis, which was also the cause of death.

Figure 1.
Figure 1. Moderately
Moderatelydifferentiated
differentiatedneuroendocrine
neuroendocrine carcinoma.
carcinoma. Tumor
Tumorbeneath thethe
beneath surface epithelium,
surface composed
epithelium, of uni-
composed of
Figure 1. Moderately
form cell, differentiated
with rare mitoses neuroendocrine
(A and B). carcinoma.staining
Immunohistochemical Tumor for
beneath the surface
chromogranin epithelium,
(C), composed
synaptophysin of uni-
(D), INSM1
uniform cell, with rare mitoses (A,B). Immunohistochemical staining for chromogranin (C), synaptophysin (D), INSM1 (E),
form cell,Ki-67
(E), and with(F).
rareMagnification
mitoses (A and
10 ×B).
10Immunohistochemical staining for chromogranin (C), synaptophysin (D), INSM1
(A and C), 10 × 20 (B, D–F).
and Ki-67 (F). Magnification 10 × 10 (A,C), 10 × 20 (B,D–F).
(E), and Ki-67 (F). Magnification 10 × 10 (A and C), 10 × 20 (B, D–F).

Figure 2. Large cell poorly differentiated neuroendocrine carcinoma. Tumor islands be-neath the surface epithelium, with
Figure
rosette 2.
Figure 2. Large
Largecell
formation,cellpoorly
composed
poorlydifferentiated
differentiatedneuroendocrine
of larger cells, carcinoma.
with many mitoses
neuroendocrine Tumor
(A and
carcinoma. B). islands be-neath
Immunohistochemical
Tumor islands the surface
be-neath epithelium,
staining
the INSM1with
for epithelium,
surface (C),
rosette formation,
synaptophysin composed
(D), p16 (E), of
and larger
Ki-67 cells,
(F). with many mitoses
Magnification 10 × 10(A
(Aand
andB).
C),Immunohistochemical
10 × 20 (B, D–F). staining for INSM1
with rosette formation, composed of larger cells, with many mitoses (A,B). Immunohistochemical staining for INSM1 (C), (C),
synaptophysin (D), p16 (E), and Ki-67 (F). Magnification 10 × 10 (A and C), 10 × 20 (B, D–F).
synaptophysin (D), p16 (E), and Ki-67 (F). Magnification 10 × 10 (A,C), 10 × 20 (B,D–F).
Cancers 2021, 13, 4813 6 of 16

Table 2. Characteristics of patients, tumors and treatment.

Patients Tumors PET-CT Therapy Outcome

Sex/Age Smoking ChT RT DFI Salvage Status
No. Site TNM Grade/ Type (Yes/No) Modality Surgery Recurrence
(Years) Status (Drug/Cycles) (Gy/fx) (mos) Therapy (Months)
Larynx
Former/
1 M/64 SG pT1cN0, M0 WD No S TLM 46+ No NED
15 py
Former/ SGL L:60/30 DOD
2 M/60 SG pT2pN2B, M0 ECE+ MD No S→RT 12 Skin S, ChT, RT
15 py ND(l) R:64/32 (26)
SGL NED
3 F/59 No SG pT1pN0, M0 MD No S 79 Neck node S
ND(b) (109)
L:70/35 Gallblader, S DOD
4 M/60 No SG cT2cN0, M0 MD No cCRT CP/5 7
R:56/35 skin ChT (38)
5 F/87 No SG pT2cN0, M0 MD Yes S T 6+ No NED
Active/ SGL L:52/26 DOC
6 F/64 SG pT2pN1, M0 PD/LC No S→RT 0 No
26 py ND(b) R:52/26 (3)
Former/ L:70/35
7 M/72 SG cT3cN0, M0 PD/LC No RT 114 No DOC
20 py R:50/25
SGL L:60/30 DOD
8 M/74 No SG pT1pN2B, M0 ECE+ PD/LC No S→RT 8 Liver, lung ChT
ND(r) R:64/32 (13)
Active/ L:70/35
9 F/26 SG cT2cN0, M0 PD/SC Yes cCRT CP + E/3 80+ No NED
10 py R:56/35
Active/ SGL
10 F/62 SG pT1pN0, M0 PD/LC + SCC Yes S 60+ No NED
40 py ND(b)
Former/ TLM L:60/30
11 M/74 SG pT2pN2C, M0 ECE+ PD/LC No S→cCRT CaP/5 5,5 No DOC
10 py ND(b) R:63/30
Former/ L:70/35
12 M/71 SG cT3cN0, M0 PD/SC Yes cCRT CP/7 27+ No NED
60 py R:56/35
Nasopharynx
CP + E/3 L:70/35 DOD
13 M/67 No NP cT4cN0, M0 MD No iC→cCRT 47 Brain RT
CP/5 R:50/25 (57)
L:70/35
14 M/80 Active/n.s. NP cT3cN0, M0 PD/LC Yes RT 12 No DOC
R:56/35
Active/ CP + E/3 L:70/35 DOD
15 M/54 NP cN2cN3, M0 PD/SC Yes iC→cCRT 7,5 Bone marrow No
45 py CP/6 R:70/35 (8,5)
Oropharynx
T(l) DOC
16 M/67 Active/n.s. OP pT1pN1, M0 PD/LC No S 32 No
ND(l) (32)
Former/ CP + E/4 L:70/35
17 M/64 OP cT4AcN2A M0 PD/LC No iC→cCRT 139+ N0 NED
20 py CP/6 R:70/35
Cancers 2021, 13, 4813 7 of 16

Table 2. Cont.

Patients Tumors PET-CT Therapy Outcome

Sex/Age Smoking ChT RT DFI Salvage Status
No. Site TNM Grade/ Type (Yes/No) Modality Surgery Recurrence
(Years) Status (Drug/Cycles) (Gy/fx) (mos) Therapy (Months)
Hypopharynx
Active/ pPH L:60/30 Liver, spleen, DOD
18 M/55 HP pT3pN2B, M0 ECE+ PD/SC No S→RT 10 N0
35 py ND(l) R:64/32 retroperitoneal (10)
Active/ L:70/35 DOD
19 M/53 HP cT3cN3, M1/lung PD/SC No iC→RT CP + E/4 6 Lung ChT
40 py R:70/35 (12)
L:70/35 DOD
20 M/59 Active/n.s. HP cT3cN3, M0 PD/SC No cCRT CP/6 0 Residual tumor No
R:70/35 (5)
No.—number; M—male; F—female; py—pack-year; SG—supraglottis; NP—nasopharynx; OP—oropharynx; HP—hypopharynx; WD—well differentiated; MD—moderately differentiated; PD—poorly
differentiated; LC—large cell; SC—small cell; S—surgery; RT—radiotherapy; cCRT—concurrent hemoradiotherapy; iC—induction chemotherapy; TLM—transoral laser microsurgery; SGL—supraglottic
laryngectomy; ND—nodal dissection; T—tumorectiomy, pPH—partial; pharyngectomy; (l)—left; (b)—bilateral; (r)—right; ChT—chemotherapy; CP—cisplatin; E—etoposide; CaP—carboplatin; L—locally;
R—regionally; DFI—disease-free interval; mos—months; NED—no evidence of disease; DOD—died of disease; DOC—died of other causes. One Patient received 26 of the planned 28 fractions of postoperative
radiotherapy due to the development of bacterial sepsis, which was also the cause of death.
Cancers 2021, 13, 4813 8 of 16
Cancers 2021, 13, x 7 of 15

Figure 3.
Figure 3. Small
Small cell
cell poorly
poorly differentiated
differentiated neuroendocrine
neuroendocrine carcinoma.
carcinoma. Tumor beneath the
Tumor beneath the surface
surface epithelium,
epithelium, composed
composed of
of
small hyperchromatic cells, with extensive crush artifacts (A and B). Immunohistochemical staining for CD56 (C), INSM1
small hyperchromatic cells, with extensive crush artifacts (A,B). Immunohistochemical staining for CD56 (C), INSM1 (D),
(D), synaptophysin (E), and Ki-67 in almost all tumor cells (F). Magnification 10 × 10 (A and C), 10 × 20 (B, D–F).
synaptophysin (E), and Ki-67 in almost all tumor cells (F). Magnification 10 × 10 (A,C), 10 × 20 (B,D–F).

3.2. Treatment
3.2. Treatment
Ten patients had upfront surgery. Five also had postoperative (chemo)RT, and two
Ten patients had upfront surgery. Five also had postoperative (chemo)RT, and two of
of these (Cases 1 and 5) received no elective treatment to the neck. The other half received
these (Cases 1 and 5) received no elective treatment to the neck. The other half received
primary RT, either alone (N = 2) or in conjunction with ChT (N = 8).
primary RT, either alone (N = 2) or in conjunction with ChT (N = 8).
In all cases, RT was conventionally fractionated (2 Gy/fraction, at five frac-
In all cases, RT was conventionally fractionated (2 Gy/fraction, at five fractions/week),
tions/week), using a 6-MV linear accelerator photon beam and CT-based computer plan-
using a 6-MV linear accelerator photon beam and CT-based computer planning. Postopera-
ning. Postoperatively, 60 Gy was delivered to the tumor bed and 64 Gy, to nodal metasta-
tively, 60 Gy was delivered to the tumor bed and 64 Gy, to nodal metastases with confirmed
ses with confirmed extracapsular tumor spread, except in Patient 6, for whom the RT was
extracapsular tumor spread, except in Patient 6, for whom the RT was prematurely termi-
prematurely terminated due to bacterial sepsis. In definitive settings, macroscopic disease
nated due to bacterial sepsis. In definitive settings, macroscopic disease was irradiated
was irradiated with 70 Gy and elective nodal regions were irradiated to an equivalent
with 70 Gy and elective nodal regions were irradiated to an equivalent dose of 50 Gy. None
dose
of theofpatients
50 Gy. None of the
received patients received
prophylactic prophylactic cranial irradiation.
cranial irradiation.
A
A concurrently
concurrently administered
administered ChTChT regimen
regimen consisted
consisted ofof 5–7
5–7 weekly
weekly applications
applications (me-
(me-
dian: 6) of cisplatin at 30 mg/m 2 (N = 2) or 40 mg/m2 (N = 4) or carboplatin at 1.5 AUC (N
2 2
dian: 6) of cisplatin at 30 mg/m (N = 2) or 40 mg/m (N = 4) or carboplatin at 1.5 AUC
=(N1);=one patient
1); one received
patient threethree
received cycles of cisplatin–etoposide
cycles combined
of cisplatin–etoposide with ChT
combined with (cisplatin
ChT (cis-
at 75 mg/m
platin at 75 mg/m , Day 1; etoposide at 100 mg/m , Days 1–3). In four patients, definitive
2, Day2 1; etoposide at 100 mg/m2, Days 2 1–3). In four patients, definitive
(chemo)RT
(chemo)RT was was preceded
preceded byby three or four
three or four cycles
cycles of
of cisplatin–etoposide
cisplatin–etoposide induction
induction ChT
ChT (the
(the
same
same regimen
regimen as as above).
above).

3.3. Immunohistochemical
Immunohistochemical and
and Molecular
Molecular Analyses
Results
Results of
ofthe
theimmunohistochemical
immunohistochemicaland
andmolecular analyses
molecular areare
analyses presented in Table
presented 3. 3.
in Table
3.3.1. Markers for Diagnosis
3.3.1. Markers for Diagnosis
Chromogranin and synaptophysin were positive in 14 (70%) and 17 (85%) tumors,
Chromogranin and synaptophysin were positive in 14 (70%) and 17 (85%) tumors,
respectively, whereas CD56 was positivity confirmed in all the analyzed tumors (100%)
respectively, whereas CD56 was positivity confirmed in all the analyzed tumors (100%)
(Figures 1C,D, 2C,D and 3E). In 17 tumors, immunohistochemical staining for all three
(Figures 1C,D, 2C,D and 3E). In 17 tumors, immunohistochemical staining for all three
traditional markers was performed. Eleven of seventeen NECs (64.7%) were positive for
traditional markers was performed. Eleven of seventeen NECs (64.7%) were positive for
all three markers; three tumors each stained positive for two or one of the markers (17.7%).
all three markers; three tumors each stained positive for two or one of the markers (17.7%).
All the studied NECs were positive for INSM1 (Figures 1E, 2C and 3D).
All the studied NECs were positive for INSM1 (Figures 1E, 2C and 3D).
Cancers 2021, 13, 4813 9 of 16

Table 3. Results of immunohistochemical and molecular analyses.

PD-L1 Mitoses
Patient’s No. Sex/Age (Years) Grade/Type Chromogranin Synaptophysin CD56 INSM1 Ki-67 p16 HPV
(CPS) (per 10 hpf)
Larynx
1 M/64 WD + + + + <2% - - Not done 1 1
2 M/60 MD + + Not done 1 + 5% + - 0 3
3 F/59 MD + + + + 5% + - 0 9
4 M/60 MD + + Not done 1 + 15% + - 0 6
5 F/87 MD + + + + 5% - - 3 3
6 F/64 PD/LC + + Not done 1 + 20% + - 0 95
7 M/72 PD/LC - - + + 20% - - 0 Not possible 2
8 M/74 PD/LC + + + + 70% + + 0 33
9 F/26 SC/SC - + + + 100% + + 0 Not possible 2
10 F/62 PD/LC + SCC + + + + 80% + - 5 40
11 M/74 PD/LC + + + + 90% + - 0 30
12 M/71 PD/SC - + + + 90% - - 0 Not possible 2
Nasopharynx
13 M/67 MD + + + + >2% - - 0 1
14 M/80 PD/LC + + + + 30% + - 0 20
15 M/54 PD/SC + + + + 70% + - 0 28
Oropharynx
16 M/67 PD/LC + + + + 70% - - 0 120
17 M/64 PD/LC - - + + 80% + - 1 25
Hypophyrynx
18 M/55 PD/SC + + + + 80% + - 0 29
19 M/53 PD/SC - - + + 80% + - 0 30
20 M/59 PD/SC - + + + 40% - - 0 21
HPV was assessed by in situ hybridization for mRNA E6/E7 HPV. In situ hybridization for EBV was performed in 3 patients with tumors in the nasopharynx; it was negative in all. Cytokeratin was positive in all
cases. In situ hybridization for HPV 6/11 was negative in all cases. No.—number, M—male; F—female; WD—well differentiated; MD—moderately differentiated; PD—poorly differentiated; LC—large cell;
SC—small cell; SCC—squamous cell carcinoma; CPS—combined positive score; hpf—high power field. 1 Not done because no tissue was left. 2 Not possible to assess number of mitoses because of crush artifacts.
Cancers 2021, 13, 4813 10 of 16

3.3.2. Proliferative Markers

The Ki-67 tumor positivity ranged from <2% to 100% (median: 55%) (Figures 1F, 2F and 3F),
and the mitosis number (per 10 high-power fields; not assessed in three PD tumors) ranged
from 1 to 120 (median: 25). In WD and MD NECs, the percentage of Ki-67-positive cells
was significantly lower than it was in PD tumors. A high degree of statistical correlation
was found between the two proliferative markers (p < 0.01; RS = 0.75; 95% confidence
interval (CI), 0.41–0.91). Ki-67 was particularly useful in small cell PD NECs in which
mitotic figures were difficult to count due to crush artifacts (Figure 3F).

3.3.3. Viral Markers

Fourteen tumors (70%) were p16-positive, but only two of them (12.5%) were found
to be positive for high-risk HPV (Figure 2E). All of the p16-negative tumors were also HPV-
negative. Of two oropharyngeal NECs, one was p16-positive, while both were negative
for high-risk HPV. None of the three nasopharyngeal NECs exhibited a positive reaction
for EBV.

3.3.4. PD-L1
CPS was determined in 19 tumors, 3 of which had a CPS ≥ 1.

3.4. Treatment Outcome and Survival

Following the initial therapy, permanent local control was achieved in 17 (85%) treated
tumors. One patient did not complete the primary treatment as planned (Case 6), and
another patient (Case 20) with a PD NEC of the hypopharynx was left with a residual
tumor upon the completion of concurrent chemoradiation (cCRT). In Patient 3, salvage
neck dissection was successfully performed 79 months after surgery for an MD supraglottis
NEC (without elective treatment to the neck) for an isolated regional recurrence, yielding
an overall rate of ultimate local control of 90% (18/20).
Systemic metastases were diagnosed in seven patients (35%; MD NEC, 3; PD NEC, 4):
in one patient at initial diagnosis and in six patients during the course of the disease
(7 to 47 months after diagnosis; median: 9). All these patients had salvage treatment
with different combinations of ChT, surgery, and/or RT and died of the disease within
0 to 31 months (median: 10) after metastatic spread was diagnosed.
Seven (35%) patients were alive and presented no evidence of disease on the close-
out date, from 6 to 139 months (median: 60 months) after the diagnosis of the NEC: one
patient with a WD tumor, two with MD tumors, and four with PD tumors. Eight (40%)
patients died of the disease at 5–57 months (median: 12.5 months) post-diagnosis; systemic
metastases were the cause of death in seven patients, and an uncontrolled primary tumor
was the cause in one. Five (25%) deaths were due to intercurrent diseases that occurred
within 3 to 114 months (median: 12 months) of NEC diagnosis. At the time of death, all
these patients had no signs of active disease, including the one who died of sepsis during
postoperative RT (Case 6).
The actuarial survival rates, with 95% CIs, at 2 and 5 years were, respectively, as
follows: LFFS: 90% (77–100); DMFS: 63% (39–86) and 52% (25–79); NEC-RFS: 56% (34–79)
and 47% (22–72); DSS: 77% (57–97) and 47% (20–74); OS: 64% (42–85) and 34% (12–57).
Upon univariate survival analysis, only the nodal stage and overall stage of the disease
proved to be statistically important factors for predicting the DMFS (at 2/5 years, Stage N0
vs. N+: 89/71% vs. 29/29%, p = 0.04; Stage I–III vs. IV: 89/89% vs. 29/14%, p < 0.01), NEC-
RFS (at 2/5 years, Stage N0 vs. N+: 89/71% vs. 23/23%, p = 0.02; Stage I–III vs. IV: 81/81%
vs. 25/13%, p = 0.03), DSS (at 2/5 years, Stage N0 vs. N+: 100/69% vs. 51/25%, p < 0.01;
Stage I–III vs. IV: 100/83% vs. 51/13%, p < 0.01), and OS (at 2/5 years, Stage N0 vs. N+:
89/61% vs. 40/10%, p < 0.01; Stage I–III vs. IV: 81/58% vs. 44/11%, p = 0.06). In addition,
LC-NEC showed a tendency toward improved DSS compared to SC-NEC (at 2/5 years:
100/80% vs. 33/33%, p = 0.06). None of the patient-, disease-, or treatment-related factors
tested affected locoregional control (Table S1).
Cancers 2021, 13, 4813 11 of 16

In 11 patients with laryngeal MD or PD NEC, the corresponding rates at 2 and 5 years

(and 95% CIs) were as follows: LFFS: 91% (74–100), DMFS: 62% (29–96), NEC-RFS: 57%
(25–89), DSS: 87% (65–100) and 60% (24–96), and OS: 72% (44–99) and 49% (17–81). In
this subgroup, the node negative neck status (Stage N0 vs. N+) also predicted improved
2/5 year DMFS (83/83% vs. 0/0%, p = 0.07), NEC-RFS (83/83% vs. 0/0%, p = 0.03),
DSS (100/80% vs. 100/0%, p < 0.01) and OS (100/80% vs. 50/0%, p < 0.01), but not
LFFS (Table S2). Neither grade (MD vs. PD) nor any of the other factors tested showed a
statistically significant effect on the studied survival endpoints

4. Discussion
Due to its rarity, the available information on HN-NEC is limited to case reports
or small series, which reflects the poor experience of clinicians with this disease. The
present report on 20 patients with 12 laryngeal and 8 pharyngeal NECs is one of the
largest single-institution series to date [21]. The central review of histological samples and
extensive histopathological and molecular characterization of tumors minimized the risk of
misdiagnosis and helped to clarify diagnostic and therapeutic dilemmas in this intriguing
malignancy. In this context, despite its single-institutional and retrospective nature, the
present study may represent solid ground on which relevant conclusions to be drawn.
The recognition of neuroendocrine differentiation is crucial for distinguishing NEC
from squamous cell carcinoma of the head and neck. Due to inherent biological and clinical
differences between the two groups of tumors, correct histological diagnosis has both
therapeutic and prognostic implications [4]. The utility of traditional markers for the
documentation of neuroendocrine differentiation, i.e., synaptophysin, chromogranin, and
CD56, is limited due to several drawbacks: the imperfect sensitivity of only 50–80% (indi-
vidual or combined) for high-grade NECs; cross-reactivity and, consequently, expression in
other, non-neuroendocrine tumors of the head and neck; and the difficulty of interpreting
the weak/focal reactivity of granular cytoplasmic staining. In addition, calcitonin is also
common, though a non-specific marker of laryngeal NECs. Its secretion from laryngeal
neuroendocrine cells is based on the embryological connection between a mesenchymal
origin of the larynx and migration of the neural crest cells that later develop the charac-
teristics of C cells. Furthermore, in rare cases, these patients even exhibit elevated serum
levels of calcitonin, which presents a challenge in the differential diagnosis of medullary
thyroid carcinoma [22]. On the other hand, INSM1 nuclear staining was found to be
easier to interpret, with a superior 99% overall sensitivity across different neuroendocrine
tumors of the head and neck and, specifically, with 95.8% sensitivity for PD NECs such as
SC-NEC [12]. To the contrary, only 2.4% of non-neuroendocrine tumors stained positive for
INSM1 in the study of Rooper et al., mainly histological types that showed reactivity with
other neuroendocrine markers [12]. Our experience confirms a high prevalence of INSM1
positivity in laryngeal and pharyngeal NECs which corroborates previous findings that
proposed its use as a first-line and stand-alone marker of neuroendocrine differentiation
for head and neck tumors [12].
HN-NECs are rare malignancies, so the treatment recommendations are based on the
results of small retrospective series or case reports published over several decades [10,11].
Due to the development of diagnostic and therapeutic methods and changes in the histolog-
ical classification of these tumors over time, the survival results reported in pooled analyses
should be interpreted with caution [3–9]. Our series is one of the largest published to
date, although it includes only 20 patients treated over a 26-year period [21]. As proposed
elsewhere, the incidence of NECs in Slovenia is negligible: these tumors represented only
0.43 and 0.17% of all the laryngeal and pharyngeal primary tumors diagnosed within the
studied period [20]. Regardless of the treatment type and tumor grade, the locoregional
control at 5 years post-therapy was excellent (90%), with only three failures: one was a
non-responder to cCRT with an extensive cN3 neck mass (Case 20); the other was a late
isolated regional recurrence successfully salvaged with surgery (Case 3); and the third was
a case after R0 surgery who died of fatal sepsis during adjuvant radiotherapy (without any
Cancers 2021, 13, 4813 12 of 16

known disease, Case 6). As expected, the most common cause of death in our group was
distant metastases. They occurred in six patients after the completion of primary treatment,
in comparable proportions in the groups of MD (3/5) and PD (3/13) tumors (p = 0.27), and
one patient had M1 disease at diagnosis (Case 19), resulting in no difference in outcomes
between the two histologic groups. The 5-year DSS and OS in our series were 47 and 34%
and did not differ with respect to the histopathological grade (WD + MD vs. PD), the
primary tumor site (larynx vs. pharynx), or the treatment modality used (primary surgery
vs. non-surgery) (Table S1). Given their wide 95% CIs, these figures are comparable to what
has been reported in systematic reviews and cancer registry data analyses and pointed to
a generally worse survival of HN-NECs compared with their squamous cell carcinoma
counterparts [1,2,11,23,24].
In the present study, half of the patients were treated with upfront surgery, and
the other half received definitive (chemo)RT. The surgical cases more often had smaller
(T1-2) primary tumors (p = 0.02). Four of these patients with MD/PD NECs received
no adjuvant (chemo)RT but remained free of disease at 6+ to 109+ months post-surgery.
On the other hand, all but two elderly patients from the RT group had one form of ChT
combined with irradiation, usually weekly platinum-based applications for the purpose of
radiosensitization. High-dose induction or concurrent ChT (cisplatin/etoposide) directed
to potential (micro)metastases was administered in five patients without obvious benefit, as
systemic metastases developed/progressed during follow-up in three of them. Based on the
above observations, we can only confirm that, besides in WD tumors, local treatment alone
also has curative potential in early-stage HN-NECs of higher grades, assuring favorable
disease control above clavicles and survival as already described by others [11,14,24].
However, an aggressive multimodal approach is needed for advanced disease, particularly
in cases with extensive neck involvement, although no survival advantage of any of the
treatment approaches (upfront surgery vs. non-surgery) was confirmed among our patients
upon univariate analysis. An obvious diversity of the clinicopathologic features between
the two therapeutic groups could play a role. Some large series also showed a lack of
differences between various treatment scenarios in NEC [11,25].
The only prognosticators that were consistently of statistical significance for all the
studied survival endpoints except the LFFS were the N-stage and the overall disease stage.
The presence of neck metastases and Stage IV disease predicted worse survival outcomes:
they affected not only the occurrence of distant metastases, which inevitably lead to patient
death, but also predicted worse OS (which reflects both cancer- and non-cancer-related
deaths). Comparable conclusions have also been reached in other, larger studies [11,24,25].
Currently, no effective systemic therapy is available: as mentioned above, three out of
five of our patients who received cisplatin/etoposide ChT died of distant metastases.
Though an encouraging initial effect of platinum-based ChT was found by Baker et al., the
responses were typically not durable, and 80% of their patients ultimately succumbed to
distant metastases [14]. According to the literature, a median progression-free survival
of 4–9 months and an OS of 10–19 months can be expected in extensive/recurrent ex-
trapulmonary PD NEC after cisplatin/etoposide-base ChT [15,26]. Unfortunately, other
chemotherapeutic agents as well as specific schedules (e.g., metronomic ChT) employed in
advanced/progressive NECs yield similarly unsatisfactory outcomes [27–30].
As an alternative, PD-1/PD-L1 checkpoint inhibitors, which are playing an increas-
ingly important role in the treatment of metastatic squamous cell carcinoma of the head
and neck, also appear to show some activity in a PD NEC, particularly when used in
combination [18,31–34]. The PD-L1 expression in our patients and in the study by Bahr
et al. was negligible [35]; however, as demonstrated by Ozdirk et al. in a small series of
neuroendocrine neoplasms of different origin but also in other histological types, patients
with negative or low PD-L1 expression on tumor cells may still derive some clinical ben-
efit from anti-PD-L1 treatment [34,36]. Additional information is expected from studies
exploring potential therapeutic strategies based on shared genomic alterations of NECs
irrespective of their sites of origin [37].
Cancers 2021, 13, 4813 13 of 16

The importance of p16/HPV status for the prediction of treatment response and
prognosis in patients with PD NEC is uncertain. The results of p16 IHC and HPV status
determination showed that, indeed, most (but not all) p16-positive and HPV-positive
cases originated from the oropharynx; however, in contrast to the case for squamous cell
carcinoma, the p16 status in PD NEC cannot be used as a surrogate marker of HPV status
due to its low specificity [38–40]. Namely, the frequently seen overexpression of p16 in
LC/SC-NEC of the head and neck may also be due to a virus-independent Rb pathway
dysregulation [41]. Our results, with a p16 positivity of 70% and only two HPV-positive
tumors (both were laryngeal primary tumors, one of them was MD NEC), corroborate
these findings. Regarding the prognostic role of HPV, Benzerdjeb et al. recently reported
an association between the involvement of HPV and improved OS in a pooled series
of 78 cases of a PD HN-NEC [42]. Since the extent of the disease was not included in
the multivariate model which, in several analyses (including ours), has proved to be an
important prognostic factor, the results of this study should be interpreted with caution.
Furthermore, upon reviewing the literature specifically focused on oropharyngeal PD NEC,
Sinno et al. found no survival advantage of HPV positivity, although without regard to the
stage of disease [40].
The prognostic importance of EBV in NECs is even less established than that of HPV.
Cai et al. reported on 12 cases of nasopharyngeal NEC, 9 being PD tumors [43]. Among
four tumors tested for EBV, one SC-NEC was negative, and three LC-NECs were positive,
with favorable responses to chemoRT observed in two of them. An additional EBV-negative
case of SC-NEC of the nasopharynx was reported by Mesonella et al. [44]. In the present
series, the EBV testing was limited to three nasopharyngeal tumors, and all were negative:
an MD NEC case, an SC-NEC case who developed systemic metastasis early, and an LC-
NEC case with complete tumor clearance after RT and who died free of disease 12 months
post-therapy. Despite favorable responses to chemoradiation in three of four reported
EBV-positive LC-NECs (3 literature cases and Case 14), the small number of EBV-tested
cases and limited/unknown follow-up information do not allow conclusions about the
prognostic role of EBV in nasopharyngeal LC-NEC to be drawn [43].
An interesting observation from the present study is the high degree of correlation
between immunohistochemical Ki-67 positivity and the number of mitoses in tumor cells. In
doubtful cases or when, for technical reasons, the number of mitoses cannot be determined,
a substitute marker of mitotic activity, which is one of the criteria for distinguishing between
PD and other types of NEC, would be desirable [9]. In this context, further studies on the
relationship between the Ki-67 labeling index and the mitosis number are warranted. In
addition, a better response to the immune-checkpoint blockade was found to be associated
with a higher proliferation rate [34].
Our study has weaknesses, which are mostly due to its retrospective nature and
limited number of studied cases. Due to the retrospective design, it is possible that the
incidence of NEC in the population is higher than that calculated; the initial histological
classification of individual cases could be incorrect, and these cases were therefore not
properly registered in the Cancer Registry database. During the 26-year period, tremendous
progress has been made, especially in diagnostics and RT treatment, which is inevitably
reflected in the differences in the quality of the management of patients diagnosed and
treated at different times. However, the small number of patients did not allow the im-
plementation of multivariate statistics that would take into account the differences in the
distribution of prognosticators across groups being compared. Another drawback to con-
sider is the limited amount of tissue for some patients, which meant that certain analyses
could not be performed. Moreover, a few biopsies were over 20 years old, with a possible
antigen degradation, which could have influenced our immunohistochemical analyses,
particularly those with a nuclear reaction, e.g., Ki-67 [45]. Finally, we did not address
certain relevant topics, such as the incidence and prognostic significance of paraneoplastic
syndromes in NEC patients, the issue of mixed tumors, and prophylactic brain irradiation
(PBI) [46–50]. Regarding the last, none of our patients had PBI: only in patients with exten-
Cancers 2021, 13, 4813 14 of 16

sive MD nasopharyngeal NEC did brain metastases occur 47 months after chemoradiation,
supporting the view that PBI does not appear to play a role in this disease [50].

5. Conclusions
HN-NEC is a rare disease, representing a diagnostic and therapeutic challenge. Im-
munohistochemical testing to INSM1 was shown to be highly prevalent in this series of
laryngeal and pharyngeal NECs. This is in line with previous findings, proposing that it
can replace the traditional panel approach for the recognition of neuroendocrine differenti-
ation. With the exception of WD tumors and early-stage MD/PD tumors, in which local
treatment ensures a high degree of disease control and good long-term survival outcomes,
others require aggressive multimodal treatment, especially in the presence of extensive
neck disease. Due to the propensity to distant spread, early and effective systemic therapy
is crucial. However, an optimal systemic agent as well as a therapeutic scenario remain to
be determined. The statuses of p16, HPV, and EBV (in nasopharyngeal primary tumors)
seem to bear no prognostic information. Pooled multi-institutional analyses, retrospective
and prospective, are warranted to properly address the existing dilemmas.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/cancers13194813/s1, Table S1: Univariate analysis of survival, all patients (N = 20), Table S2:
Univariate analysis of survival, moderately and poorly differentiated neuroendocrine carcinoma of
the larynx (N = 11).
Author Contributions: Conceptualization, P.S. and N.Z.; methodology, P.S. and N.Z.; validation,
A.F. and G.P.; formal analysis, P.S.; data curation, P.S., R.Š., C.G.-K., B.L. and N.Z.; writing—original
draft preparation, P.S. and N.Z.; writing—review and editing, R.Š., A.F., C.G.-K., B.L. and G.P.;
project administration, P.S. and A.F. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Slovenian Research Agency (ARRS), grant numbers
P3-0307 and P3-0054.
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki, and the study protocol was approved by the Committee for Medical
Ethics and the Protocol Review Board of the Institute of Oncology Ljubljana (ERIDEK-0076/2020,
19 November 2020).
Informed Consent Statement: For retrospective studies, a written patient consent is deemed unnec-
essary according to national and institutional regulations.
Data Availability Statement: The datasets analyzed during the current study are available from the
corresponding author on reasonable request.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.

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