Invasive stratified mucin-producing carcinoma: a clinicopathological analysis of three cases

ABSTRACT

Objective: To investigate the clinicopathologic features and immunophenotype of invasive-stratified mucin-producing carcinoma (ISMC).

Methods: We retrospectively analyzed three patients who underwent surgery for pathologically confirmed ISMC from January 2017 to December 2017 in Anyang Tumor Hospital. The pathological features, immunophenotype, and prognosis were discussed.

Results: Clinical symptoms and imaging were atypical. Microscopically, the arrangement of tumor cells is close to that of squamous cell carcinoma, showing flake or nested growth. Morphologically, tumor cells were reminiscent of adenocarcinoma. The tumor cells showed abundant intracytoplasmic mucus which routinely created spacing between adjacent nuclei. The nuclei was round or oval with irregular karyotypes, thick nuclear membrane, staining empty light, rough chromatin, visible small nucleoli. Mucous vacuoles or pink-stained foam were visible in the cytoplasm. Mitotic features and apoptotic bodies were seen in the invasive component of all three cases, whereas all but one of these showed a neutrophil-dominant inflammatory cells infiltration. A large number of histocytes were found in the stroma of two cases. Special staining of mucus confirmed the presence of mucus in cytoplasm. Immunohistochemical results showed that tumor cells were strongly and diffusely positive for p16 and CK7 and had high Ki-67 expression.

Conclusions: ISMC is a rare tumor in female genital tract and is often misdiagnosed as squamous cell carcinoma and other cervical cancers. The diagnosis mainly depends on the unique clinicopathologic features together with the immunophenotype. Treatment and prognosis, like other cervical cancer, are mainly based on clinical stages.

1. Introduction

Stratified mucin-producing intraepithelial lesion (SMILE) is a rare columnar cell cervical neoplasm, first described by Park et al.¹ in 2000 and the recent 2014 World Health Organization classification² described SMILE as a cervical glandular premalignant lesion, which originated from embryonic cells at the transformation zone by transdifferentiation during high-risk HPV-associated carcinogenesis. It is characterized by stratified, immature epithelial cells displaying varying quantities of intracytoplasmic mucin throughout the majority of the lesional epithelium. Lastra et al.³ recently reported the similarity in appearance between invasive components and SMILE and proposed the invasive form of SMILE to be “invasive stratified mucin-producing carcinoma” (ISMC). Because of these limited data, this review discusses the clinicopathologic features, immunohistochemical diagnosis and differential diagnosis of ISMC in order to improve the understanding of the tumor.

2. Materials and methods

The clinical and pathological data of three patients with ISMC diagnosed by pathology department of Anyang Tumor Hospital from January 2017 to December 2017 were retrospectively analyzed and all cases were confirmed by at least two senior pathologists. During the same period, there were 165 cases of cervical carcinoma, including 143 cases of squamous cell carcinoma, 21 cases of adenocarcinoma and 1 case of adenosquamous carcinoma.

The specimen was fixed in 4% buffered formalin, routinely processed, with tissue sections embedded in paraffin. The sections were cut at 4μm in thickness and were stained with hematoxylin and eosin (H&E). Immunohistochemistry was performed according to standard protocols. The following antibodies were used: p16 (Dako Denmark, prediluted), p63 (Dako Denmark, prediluted), CK5/6 (Dako Denmark, prediluted), CK7 (Dako Denmark, prediluted),　 MIB1/Ki67 (Dako Denmark, prediluted). All negative and positive controls were included. The specific part of the tumor cells were stained as brown and yellow, which was considered as positive expression. CK5/6 and CK7 were stained in the cytoplasm. p63, and Ki67 were stained in the nucleus. p16 was stained in the nucleus and cytoplasm. Special staining of mucus (AB-PAS) was performed according to standard protocols. The results showed that acidic mucus was blue, neutral mucus was red, and mixed mucus was purple red.

3. Results

3.1. Clinical data

For three cases of ISMC patients, the average age was 47 years old (ranged from 30 to 64 years old). They never smokes and has no history of malignancy and TB. All of them have offspring, one of them has gone through menopause. One case complained vaginal bleeding after menopause, one case complained contact vaginal bleeding for 6 months, one case was found by physical examination. Physical examination: three patients had different degrees of mass in the cervical, which was characterized by erosion, poor crisp, and positive for blood contact. Magnetic Resonance Imaging (MRI) showed that there was a soft tissue in the cervix, and no enlarged lymph nodes were found in pelvic cavity. With polymerase chain reaction (PCR), two cases were tested positive for HPV types 16 and 18, and one case was positive for HPV types 18. The biopsy of the colposcopy was diagnosed as carcinoma, of which two were adenocarcinoma and one was squamous cell carcinoma (Table 1). The preoperative clinical diagnosis is cervical carcinoma.

Table 1.

Clinicopathological findings in invasive stratified mucin-producing carcinoma.

Case no.	Age	HPV	Symptom	Preoperative diagnosis	Depth of Invasion	Postoperative diagnosis	Treatment	Follow-up(mon)
1	47	HPV18+	Physical examination	Ade	1.5cm	ISMC(one LN meta)	Hyst	Alive(27 mon)
2	64	HPV16+ HPV18+	Vaginal bleeding after menopause	SCC	1.2cm	ISMC+HSIL	Hyst	Alive(19 mon)
3	30	HPV16+ HPV18+	Contact vaginal bleeding	Ade	2cm	ISMC+HSIL	Hyst+Chemo+Rad	Alive(26mon)

Abbreviations: Ade, adenocarcinoma; SCC, squamous cell carcinoma; LN, lymph node; meta, metastasis; Hyst, hysterectomy; Chemo, chemotherapy; Rad, radiation;

3.2. Gross examination

Tumor in three cases were confined to the cervical with either nodular, ulcer or exogenous growth, and no significant metastasis out of cervical was observed. The cut surface of the tumor was a tan or gray-white part of the tumor with bleeding and necrosis.

3.3. Microscopical examination

Three cases were classified as invasive carcinoma with morphologic features identical to SMILE and distinct from those in squamous cell carcinoma or usual-type endocervical adenocarcinoma. From the arrangement point of view, the tumor cells is more similar to squamous cell carcinoma, showing flake or nested growth. From the cell itself, the characteristics of tumor cells were more similar to those of adenocarcinoma. The tumor cells showed abundant intracytoplasmic mucus (Figure 1A) which routinely created spacing between adjacent nuclei and formed the glandular cavity (Figure 1B) in some area. The amount of mucus in cells varies from case to case or from area to area (Figure 1C). The nuclei was round or oval which irregular karyotypes, thick nuclear membrane, staining empty light, rough chromatin, visible small nucleoli. Mucous vacuoles or pink-stained foam were visible in the cytoplasm. Mitotic features and apoptotic bodies were seen in the invasive component of all three cases, whereas all but one of these showed a neutrophil-dominant inflammatory cells infiltration (Figure 1D). A large number of histocyte were found in the stroma of two cases.

Figure 1.

A. The tumor cells showed abundant intracytoplasmic mucus; B. The tumor cells routinely created spacing between adjacent nuclei and formed the glandular cavity in some area; C. The amount of mucus in cells varies from case to case or from area to area; D. The tumor showed an associated neutrophil-dominant inflammatory cells infiltrate (H&E × 100 staining).

3.4. Immunophenotype

Three cases were strongly and diffusely positive for p16 (Figure 2A) and CK7 (Figure 2B), and they showed high Ki-67 (Figure 2C) expression. The immunohistochemistry of p16 showed a pattern of diffuse＇block-type＇ nuclear and cytoplasmic immunoreactivity. Three cases were negative or focally and weakly positive for p63 and CK5/6. The Ki-67 positive index was 70–80%.

Figure 2.

Immunohistochemistry and special staining of the ISMC.

A: Diffuse‘block-type’ immunoreactivity for p16.B: Diffuse, positive expression of CK7.C: High expression of Ki-67 (IHC with SP method×100).D: Mucus staining showed scattered blue staining (Special staining of mucus with AB-PAS method×100).

3.5. Special staining

Special staining of mucus in three cases showed scattered blue staining, which confirmed the presence of mucus in cytoplasm (Figure 2D).

3.6. Follow-up

Three patients received surgical treatment and were successfully followed up. All patients were healthy with no recurrence after 19–27 months’ follow-up.

4. Discussion

SMILE is an uncommon intraepithelial lesion. The initial 2000 proposal by Park et al.¹ and the recent 2014 World Health Organization classification² described SMILE as a variant pattern of AIS. The typical appearance of SMILE was a stratified epithelium similar in architecture to a high-grade CIN. However, one consistent feature distinguishing SMILEs from conventional CIN was the conspicuous spacing of nuclei in the lower to middle epithelial layers, coincident with the presence of cytoplasmic mucin.¹ Lastra et al.³ recently reported the similarity in appearance between invasive components and SMILE and proposed the invasive form of SMILE to be “invasive stratified mucin-producing carcinoma” (ISMC), which originated from embryonic cells at the transformation zone by transdifferentiation during high-risk HPV-associated carcinogenesis. It is characterized by stratified, immature epithelial cells displaying varying quantities of intracytoplasmic mucin throughout the majority of the lesional epithelium.

4.1. Clinical features

ISMC is an uncommon malignant tumor of the female cervical cancer, constituting 1.8% (3/165) of cervial carcinoma and 14.3% (3/21) of cervical adenocarcinoma that were diagnosed from January 2017 to December 2017 in our hospital. ISMC is more common in middle-aged women (30–64 years old), often with irregular vaginal bleeding and vaginal bleeding after menopause. Gross examination: three patients had different degrees of mass in the cervical, which was characterized by erosion, poor crisp, and positive for blood contact. MRI showed that there was a soft tissue in the cervix. The cut surface of the tumor was a tan or gray-white part of the tumor with bleeding and necrosis. Preoperative colposcopy biopsy can be easily misdiagnosed as squamous cell carcinoma if the physician is unfamiliar with the disease. Therefore, the routine histological sections and immunohistochemical staining of ISMC are particularly important for the diagnosis.

4.2. Diagnosis

The morphology for SMILE and ISMC is basically the same. The typical appearance of SMILE was a stratified epithelium similar in architecture to a high-grade CIN. However, one main feature distinguishing SMILE from high-grade CIN was distinct separation of nuclei by conspicuous cytoplasmic vacuoles in the lower to middle epithelial layers. The nuclei was round or oval which irregular karyotypes, thick nuclear membrane, staining empty light, rough chromatin, visible small nucleoli. Mucous vacuoles or pink-stained honeycombed were visible in the cytoplasm. Mitotic figures and apoptotic bodies were easily visible in the invasive component of all three cases. A large number of histocyte were found in the stroma and neutrophil-dominant inflammatory cells infiltrate were obvious of two cases. Tumor cells were strongly and diffusely positive for p16, CK7, and high Ki-67 expression. It is worth mentioning that due to the presence of cytoplasmic mucus, p16 immunohistochemistry was performed diffuse‘block-type’ nuclear and cytoplasmic immunoreactivity.¹ Two cases were negative for p63 and CK5/6, focal basal cell staining was observed with p63 and CK5/6 in one case. IMP3 is a member of the insulin-like growth factor protein family and is a useful maker for several carcinomas.^4–8 It has been shown that IMP3 is a useful marker of cervical AIS, whereas benign endocervical glands were negative.⁹ Boyle and McCluggage¹⁰ reported that SMILE and HSIL were completely negative for IMP3. Onishi and Sato¹¹ found that IMP3 was more strongly and diffusely positive in invasive carcinoma than intraepithelial lesion. In order to further clarify whether it is adenocarcinoma, mucus specific staining is essential. In the three patients we reported, mucus staining showed scattered blue staining, confirming the presence of mucus in cytoplasm.

4.3. Differential diagnosis

4.3.1. Adenosquamous carcinoma

The diagnosis of adenosquamous carcinoma requires the simultaneous identification of histologic pattern of adenocarcinoma and squamous cell carcinoma. Scattered mucin-producing cells may occur in a squamous cell carcinoma^12,13 and such tumors should not be labeled as adenosquamous carcinoma. Since the presence of mucin within squamous carcinomas has not been shown to have any prognostic or predictive value, routine staining for mucin in squamous carcinomas is not recommended.² Very rarely, a carcinoma dispalys three cell types (epidermoid, mucin-producing and intermediate), these tumors can be termed “muco-epidermoid” carcinomas of the cervix.¹⁴ These neoplasms were defined strictly by a squamoid growth pattern with demonstrable intracellular mucin and it is advised that this diagnosis should be confirmed by mucin staining in any tumor showing finely vacuolated cytoplasm and an absence of peripheral palisading.¹⁵ These tumors show similar features to their salivary gland, easy to cystic sometimes and have no associated intraepithelial lesions.

4.3.2. Glassy cell carcinoma

Glassy cell carcinoma is a poorly differentiated variant of adenosquamous carcinoma characterized by cells with sharp cytoplasmic margins. The tumor cells have eosinophilic cytoplasm, being characterized by cells with a moderate amount of cytoplasm having a ground glass or finely granular appearance, a distinct cell wall that stains with eosin and PAS, and enlarged nuclei with prominent nucleoli.¹⁶ Glassy cell carcinoma show a prominent eosinophilic infiltrate in the stroma surrounding the nest of neoplastic epithelium.¹⁷

4.3.3. Squamous cell carcinoma

Most carcinoma exhibit sheet-like growth and infiltration as networks of anastomosing bands or single cells with an intervening desmoplastic or inflammatory stroma. The most important difference from other tumors is that the tumor cells are polygonal, showing interbridge, keratin pearls or intracellular keratosis. Immunohistochemically, squamous cell carcinoma is positive for p63 and CK5/6.

4.3.4. Clear cell carcinoma

Clear cell carcinoma is composed of three types of cells (hobnail, flat and clear cells). Four different types of structures (solid, cystic, tubular, papillary) can be observed. High-grade nuclear atypia can be seen at least locally in most cases.

4.4. Treatment and prognosis

Clinical stage is of great significance to the prognosis and treatment. There is an obvious correlation between survival and disease stage, with 99% five-year survival for stageⅠA1, 65% for ⅡB and 43% for ⅢB.¹⁸ The higher the clinical stage, the greater the risk of death in patients with cervical cancer. Other independent prognostic factors include lymphovascular invasion, lymph node status, depth of invasion, size of tumor and the state of cutting edge. Lastra et al.³ reported three cases of ISMC that one patient died after 1.5 months and two had lung metastases after 9 months and 36 months. Onishi et al.¹¹ reported three patients of ISMC who survived after receiving hysterectomy, radiotherapy or chemotherapy. In our study, all three patients underwent hysterectomy, double attachment resection, and pelvic lymph node dissection. 2 of them received postoperative radiotherapy and chemotherapy. No recurrence was observed in the three cases.

Funding Statement

No external funding was involved during this analysis.

Conflict of interest

The authors report no conflicts of interest. The authors are responsible for the content and writing of the paper.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.