Hybrid Carcinoma of The Parotid Gland: A Report of Three Extremely Rare Cases with An Immunohistochemical Analysis and A Review of The Literature

Background: Hybrid carcinoma (HC) of the salivary glands is dened as when two or more kinds of carcinoma exist at the same location in a single mass. Herein, we report 3 rare cases of salivary gland HC. Case Presentation: Case 1 involved an 86-year-old Japanese male. Case 2 involved a 70-year-old Japanese female. Case 3 involved a 66-year-old Japanese male. Histologically, case 1 involved a combination of salivary duct carcinoma (SDC) and squamous cell carcinoma (SqCC). Immunostaining indicated that the former was positive for gross cystic disease uid protein (GCDFP)-15 and androgen receptor (AR), whereas the latter expressed cytokeratin (CK) 5/6 and p63. Case 2 involved a combination of SqCC and neuroendocrine carcinoma (NEC). Immunostaining indicated that the former was positive for CK5/6 and p40, whereas the latter was positive for synaptophysin and neural cell adhesion molecule. Case 3 involved a combination of SDC and epithelial-myoepithelial carcinoma (EMC). Immunostaining indicated that the former was positive for GCDFP-15 and AR, whereas the inner cells of the latter were positive for CK7, and the outer cells of the latter were positive for alpha-smooth muscle actin and p40. A transitional zone between the SDC and EMC existed in case 3. Conclusions: Therefore, we diagnosed them as parotid gland HC. This is the rst report of a case of HC involving a combination of NEC and SqCC. In case 3, it was speculated that high-grade SDC arose from low-grade EMC. However, as HC is different from carcinoma with high-grade transformation, HC should be diagnosed carefully.


Introduction
In 1996, Seifert and Donath described a series of 5 cases of salivary gland tumors containing two different types of neoplasms and called them "hybrid tumors" [1]. Nowadays, hybrid tumors are de ned as tumors consisting of at least two different tumor entities that develop and merge in the same topological area. Hybrid tumors account for up to 0.1% of all salivary gland tumors, and both benign and malignant hybrid tumors have been reported [1,2].

Case Presentation
We extracted 3 cases of salivary gland HC from a pathology le and one of the author's consultation les (K.K.) for the period from 1992-2019. All cases involved primary salivary gland HC. All of the slides for these cases were reevaluated, with particular attention paid to the two different histological components, the presence or absence of transitional zones, and the absence of primary neoplasms from other organs, especially in the head and neck region.

Case 1 Clinical ndings
The patient was an 85-year-old Japanese male, who had noticed a rapidly swelling mass in the left parotid region some years ago.
He was admitted to his local clinic and was diagnosed with adenocarcinoma after a ne-needle aspiration biopsy (FNAB). He was admitted to our hospital 20 months later. An immovable mass was present in the left parotid region, but the patient was not suffering any pain or facial nerve paralysis. Magnetic resonance imaging (MRI) showed an ill-de ned high intensity region and a focal low intensity region on T1-weighted imaging, and a low intensity region and focal high intensity region on T2-weighted imaging ( Figure 1A). Positron emission tomography (PET) showed accumulation in the left parotid gland mass. Under a clinical diagnosis of left parotid gland cancer (pT4aN0M0), total parotidectomy with skin and facial nerve resection was performed. No postoperative treatment was administered. There was no evidence of local recurrence or metastasis at 2 years after the operation.

Pathological ndings
Macroscopically, an ill-de ned yellowish-white mass, with partial cystic changes, and yellowish-green contents inside the cystic spaces, was seen ( Figure 1B). The tumor measured 4.0 x 3.2 x 5.3 cm.
Histologically, the tumor was composed of a combination of an SDC and an SqCC. The major component (i.e., the SDC), which accounted for 80% of the tumor, had a classical and typical histology, involving microcystic to marked cystic dilation, and exhibited Roman-bridge structures and comedonecrosis by cancer cells (Figure 1C), together with eosinophilic cytoplasm, marked nuclear pleomorphism, and a high mitotic rate. Immunohistochemical staining was performed on 4-μm-thick sections, which were cut from formalin-xed and para n-embedded tissue. The tumors were considered to be diffusely positive (+++), positive (++), partially positive (+), and focally positive (F+) when ≥50%, 20-49%, 10-19%, and 1-9% of the neoplastic cells were positive, respectively. The HER2 score was estimated according to the criteria of Wolff et al [24]. Epidermal growth factor receptor (EGFR) expression was categorized as positive or negative. According to Boyle's evaluation criteria [25], a tumor was considered to be p53positive if >50% of the tumor cell nuclei showed strong reactivity, which was considered to be indicative of a p53 point mutation. Carcinoma cells that exhibited no p53 immunostaining despite weak positivity being seen in the in ltrating lymphocytes raised a high suspicion of the deletion (i.e., loss) of p53. Weak of sparse p53 immunostaining was considered to be indicative of the presence of wild-type p53. The antibodies used in this study and the results of the immunohistochemical analysis are summarized in Table 2. Immunostaining showed that the SDC component was strongly positive for cytokeratin (CK) 7, gross cystic disease uid protein (GCDFP)-15, and androgen receptor (AR), and partially positive for EGFR and CK5/6 (Figures 1D), but negative for human epidermal growth factor receptor 2 (HER2), p63, and p40. Therefore, the SDC was subclassi ed into the apocrine B phenotype, according to Takase's classi cation of SDC [26].
At least 20% of the tumor was made up of nest-like structures composed of proliferating cancer cells, which exhibited keratinization and squamous differentiation ( Figure 1E). The cancer cells in this component displayed marked nuclear atypia, single cell keratinization, and intercellular bridges. This component was considered to be a moderately differentiated SqCC.
As both components were focally and weakly positive for p53, they were considered to exhibit the wild-type pattern. The Ki-67 indices of the SDC and SqCC components were 58% and 43%, respectively. Both components were well demarcated, but some areas showed mixed nests composed of both SDC and SqCC.
We nally diagnosed the patient with HC composed of SDC and SqCC.

Clinical ndings
The patient was a 79-year-old Japanese female, who suffered from swelling of the left parotid region and a disturbance of oral opening. She noticed a small painless mass in the left parotid region. The mass rapidly enlarged, and she was admitted to another hospital a month later. An immovable mass was seen in the left parotid region, but no lymph node swelling or facial nerve paralysis was observed. FNAB and MRI showed no malignancy at that time. After approximately 3 months, FNAB was performed again, and the lesion was cytologically diagnosed as SqCC. Computed tomography (CT) revealed an ill-de ned irregularly shaped mass in the left parotid gland, and MRI showed a region of iso-intensity on T1-weighted imaging and low intensity on T2-weighted imaging. PET only showed accumulation in the left parotid mass and did not show any other primary lesion. Under a clinical diagnosis of left parotid gland cancer (cT4aN0M0), left total parotidectomy and left mandibulectomy were performed.
Postoperative radiotherapy (66 Gy) was administered. There was no evidence of recurrence or metastasis at 7 months after the operation.

Pathological ndings
Macroscopically, a single mass was seen (Figure 2A). An examination of the cut surface of the tumor revealed an ill-de ned, yellowish-white mass. The central portion of the mass was somewhat whitish.
On the other hand, the central portion of the mass was composed of sheet-like and/or nest-like structures, containing large atypical polygonal cells, with relatively loose connections among the tumor cells ( Figure 2C). They infrequently displayed rosette-like structures, but the central necrosis was frequently seen. Immunostaining indicated that they were positive for NCAM and CK8, and partially positive for synaptophysin and INSM1 ( Figure 2D). Only a few cells were positive for chromogranin-A. On the other hand, the tumor cells were entirely negative for CK5/6, p63, and p40, which are squamous cell markers. Therefore, this component was considered to be an LCNEC.
The Ki-67 labeling indices of the SqCC and LCNEC were 51% and 68%, respectively. The border between the two components was irregular, but the components could be clearly divided via immunostaining ( Figure 2E). No obviously transitional zone was observed. As the SqCC component was strongly positive for p53 and the LCNEC component was weakly positive for p53, the SqCC displayed a mutation pattern, and the LCNEC exhibited the wild type pattern ( Figure 2F and 2G).
We nally diagnosed the patient with HC composed of SqCC and LCNEC.

Clinical ndings
The patient was a 66-year-old Japanese male, who had developed a swollen mass on the right side of his neck. As the mass enlarged rapidly and was painful when pressure was applied to it, the patient was admitted to a local clinic, and a FNAB resulted in a diagnosis of a "malignant tumor, suspected, especially, SDC". The patient was admitted to our hospital one month later. An immovable mass was present in the right parotid region, and the patient was experiencing pain and facial nerve paralysis. Under a clinical diagnosis of right parotid gland cancer (pT4aN2bM0), total parotidectomy with skin and facial nerve resection was performed. Postoperative chemoradiotherapy was administered, but the patient stopped attending follow-up examinations after 2 years.

Pathological ndings
Macroscopically, an ill-de ned yellowish-white mass, which exhibited partial lobulated growth and slight cystic changes, was seen ( Figure 3A). The tumor measured approximately 3.3 x 3.0 x 4.8 cm.
Histologically, the tumor was composed of a combination of an SDC and an EMC. The EMC component, which accounted for 55% of the tumor, exhibited the typical histology; i.e., it demonstrated a bi-phasic growth pattern, involving inner ductal cells and outer clear cells ( Figure 3B). These cells displayed mild to moderate cellular atypia, and a low mitotic rate, and apocrine differentiation of the inner ductal cells, e.g., eosinophilic cytoplasm and sprouting on the luminal side, was also observed. Immunostaining showed that the inner cells of this component were positive for EMA and CK7 ( Figure 3C) and partially positive for CK5/6, whereas the outer clear cells were positive for CK14, CK5/6, alpha-smooth muscle actin (α-SMA), p63, p40, vimentin, and Wilms' tumor (WT)-1 ( Figure 3D). The inner cells were also positive for EGFR, but they were negative for HER2. Therefore, this component was considered to be an EMC.
Approximately 45% of the tumor was composed of proliferating atypical ductal cells, which exhibited marked cellular atypia and eosinophilic cytoplasm. The tumor cells displayed the cystic-papillary, Roman bridge, and solid nest patterns, as well as partial comedonecrosis ( Figure 3E). The mitotic rate was relatively high. Immunostaining indicated that they were positive for EMA, CK7, AR, GCDFP-15, and HER2 (2+) (Figures 3F and G), but negative for EGFR and myoepithelial markers. Therefore, this component was considered to be an SDC.
A transitional zone between the SDC and EMC was observed. Atypical apocrine cells were present among the clear myoepithelial cells of the EMC component ( Figure 4). As both components were focally positive for p53, they were considered to display the wild-type pattern. The Ki-67 labeling indices of the SDC and EMC components were 23% and 18%, respectively (Supplemental gure 12A and 1B). Therefore, according to Takase's classi cation [26], the SDC component belonged to apocrine A subtype.
We nally diagnosed the patient with HC composed of EMC and SDC.

Discussion And Conclusions
Salivary gland HC is an extremely rare and unique neoplasm, and, to the best of our knowledge, only 38 cases have been reported in the English literature, as shown in Table 1 [1,. Various combinations of carcinoma have been described in HC, and SDC, EMC, and adenoid cystic carcinoma (AdCC) are frequently involved. However, as the tubular subtype of AdCC often exhibits EMClike features, such as a bi-phasic structure, involving inner ductal cells and clear outer cells [27], HC should be diagnosed carefully in cases involving a combination of EMC and AdCC. 28 HC should not be confused with collision tumors, which arise from two different adjacent tumors. Kufeld et al. described a collision tumor consisting of an AdCC of the pharynx and an SqCC of the larynx [29]. In addition, HC should be distinguished from carcinomas with bi-phasic differentiation, which are single entities composed of two different cellular types, such as EMC. Furthermore, carcinoma with high-grade transformation (HGT) also needs to be differentiated from HC [30]. Several salivary gland tumor entities that exhibit two or more different morphologies should also be ruled out before a diagnosis of HC is made. These entities include collision tumors, synchronous tumors, multiple tumors [31], carcinoma with metaplastic changes, carcinoma with HGT (dedifferentiated carcinoma) [32], and the sarcomatoid variant of SDC [33]. As all of our cases involved two distinct carcinomas in the same topological area, which had combined to produce a single tumor mass, they were de ned as HC.
The presence of a transitional zone between two carcinoma components (like in case 3) suggests that they both have the same origin; therefore, detecting such regions allowed us to differentiate our cases from collision tumors, which involve two carcinomas arising at independent topological sites and then subsequently coming into contact. It also allowed us to distinguish them from synchronous and multiple tumors. On the other hand, SDC, SqCC, and LCNEC are entirely of epithelial origin, whereas EMC is composed of both epithelial and myoepithelial cells. Therefore, the presence of a combination of SDC and SqCC (like in case 1) suggests that both components might not be of identical origin, but rather that the SqCC might have arisen through squamous metaplasia in SDC. However, we could not nd any histological or immunohistochemical evidence to support this. On the other hand, cases 2 and 3 consisted of a combination of SqCC and LCNEC, and SDC and EMC, respectively. In case 2, an intermingled area was seen between two carcinoma components, and one component might have arisen from the other. In case 3, atypical inner cells with apocrine features were frequently seen in the EMC component; thus, the SDC might have arisen from the EMC. Nagao et al. proposed that if each component occupied >30% of the tumor mass and could be separated from the other component and the proliferative activity of the two components was markedly different, HC should be considered [3]. In case 1, the SqCC component only accounted for 20% of the tumor mass. However, as the SqCC showed a typical histology and the keratinization observed in the SqCC is not seen in squamous metaplasia of SDC, we considered that this case should be diagnosed as HC. Furthermore, in HC both components seem to display similar levels of proliferative activity. If one component of a suspected HC has high proliferative activity and the other has low proliferative activity, carcinoma with HGT should also be considered [32] As both components exhibited similar levels of proliferative activity in all of our cases, we considered that they were not carcinomas with HGT. Recently, Hamamoto et al. reported a rare case in which SDC arose from EMC, and they considered that their case should be diagnosed as HC rather than carcinoma with HGT, despite the fact that the two components displayed different proliferative activities and histological grades [23]. Our case 3 might have undergone a similar tumorigenic process to their case.
Our case 2 involved a combination of SqCC and LCNEC, and no other cases involving such a combination have been reported. In the current version of the WHO tumor classi cation, LCNEC was included as a type of poorly differentiated carcinoma [34].
However, we consider that in cases that are positive for neuroendocrine markers, "neuroendocrine carcinoma, including LCNEC," rather than merely "poorly differentiated carcinoma" should be used as a pathological diagnostic term. Salivary gland LCNEC itself is extremely rare. To the best of our knowledge, only 8 cases of LCNEC have been reported in the English literature [35][36][37][38].
Salivary gland LCNEC usually exhibits a worse prognosis than other salivary gland carcinomas and a predilection for males. On the other hand, SqCC of the salivary glands is rare, and it is important to always exclude metastatic or invasive SqCC from other sites [39], such as the oral cavity, oropharyngeal region, head and neck, or skin like Merkel cell carcinoma. In case 2, there was no evidence of other primary sites on PET or other systemic examinations, and the immuno-negativity for CK20 indicated that this tumor was not Merkel cell carcinoma of the skin, and as the tumor was composed of a combination of SqCC and LCNEC, the parotid gland was considered to be the primary site in this case. This is the rst reported case of HC involving a combination of SqCC and LCNEC.
Although the previously reported cases of HC frequently showed poor outcomes , the outcomes of our cases were better; i.e., no recurrence or distant metastasis was seen. Although little information is available, several investigators have suggested that the aggressiveness of HC is determined by the histologically higher-grade component. 23 It would appear that treatments for HC should be targeted at the higher-grade component, but this requires further con rmation in much larger series with longer follow-up periods.
In conclusion, 3 HC cases, which involved combinations of SDC and SqCC, SqCC and LCNEC, and SDC and EMC, respectively, were reported, and such cases are extremely rare. Although our cases showed better prognoses than previously reported HC cases, long follow-up is needed. HC might originate as one pre-existing carcinoma and then might acquire another differentiation direction early in the carcinogenic process, producing two components in a single mass.

Consent for publication
Written informed consent for the publication of clinical details and/or clinical images was obtained from the patients. A copy of the consent form is available for review by the Editor of this journal.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests related to this study.

Funding
The authors declare that they received no funding support for this study.    The HER2 scores were determined according to the criteria developed by Wolff et al. 24 The p53 expression pattern was estimated according to the criteria developed by Boyle et al. 25

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