Oesophageal cancer composed of mixed histological types

European Journal of Surgical Oncology 1996; 22:225-231

Oesophageal cancer composed of mixed histological types Hiroyuki Kuwano, Noriaki Sadanaga, Masayuki Watanabe, Mitsuhiro Yasuda, Tadahiro Nozoe and Keizo Sugimachi Department of Surgery II, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan

Glandular or adenoid cystic differentiation and sarcomatous features are sometimes encountered in oesophageal cancer and such histological variants often coexist with ordinary squamous cell carcinoma. So far no serial evaluation of such mixed histological types of oesophageal cancer has ever been performed, we therefore clinicopathologically investigated such cases while an immunohistochemical study of the p53 protein was also done on four available cases in order to discuss the histogenesis of these turnouts. Among the 600 patients with surgically resected oesophageal cancer, seven cases were found to have a mixture of histological variants with ordinary SCC (squamous cell carcinoma) (adenocarcinoma 1, adenoid cystic cancer 3, sarcomatous component 2, basaloid cystic cancer and sarcomatous component 1). All but one case had an elevated type tumonr while the predominant components of protrusion were not SCC but variant histological types. Six tumours were restricted to the submncosal layer. An immunohistochemical study of the p53 protein revealed that two of four cases were positive in both the SCC and variant patterns. Mixed histological types of oesophageal cancer composed of SCC and other variants often occur and thus the carcinogenesis of both histological types are considered to be aspects of the same mechanism.

Key words: oesophageal cancer; mixed type of cancer; p53 protein; pseudosarcoma.

Introduction

Although the most common histological type of oesophageal carcinoma is squamous cell carcinoma, both glandular differentiation t'-' and adenoid cystic differentiation3 have also been demonstrated to be not uncommon following serial investigations of oesophageal cancer tissue specimens. Moreover, spindle cell patterns of malignant tumours, including pseudosarcoma and carcinosarcoma, also occur occasionally.4'~ Such histological variations of oesophageal carcinoma sometimes co-exist with ordinary squamous cell carcinoma in the same malignant lesions, t Moreover, the proportion of such histological variations within the cancerous area vary and they sometimes occur broadly or are located focally. A serial evaluation of such oesophageal tumours demonstrating a mixture of such histological variation with ordinary squamous cell carcinoma is thus considered to be important for investigating the histogenesis of such tumours, and also to help in suggesting the mode of origin in oesophageal cancer. Either a deletion or mutation of tumour suppresser genes is considered to be a key event in carcinogenesis. The human p53 gene is located on chromosome 17p3 and encodes a nuclear protein which may be important in the regulatory control of cell proliferationY A frequent mutation of the p53 gene at exons 5, 6, 7 and 8 has been seen in a wide Correspondence to: Hiroyuki Kuwano, MD, Department of Surgery II, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan. 0748-7983/961030225 +07 $12.00/0

variety of human cancers, including oesophageal carcinoma) Mutant p53 proteins are more stable than wild type proteins and thus their accumulation can also be detected immunohistochemically.9 Overexpression of p53 protein is commonly present in oesophageal cancer) Duhaylongsod et al. demonstrated that the prevalence of p53 protein expression in oesophageal adenocarcinoma was 80%) o thus mutation and overexpression of p53 protein is a common genetic alteration observed in oesophageal adenocarcinoma. These p53 protein accumulation was also demonstrated in the squamous cell carcinoma of the oesophagus as well as in their precancerous lesions, t° In the current study, we analysed the cases of oesophageal malignant turnouts composed of an admixture of different histological types while also performing an immunohistochemical study to detect the presence of the p53 protein in such tumours. Particular attention was directed to the mode of origin in such tumours while the histogenesis of oesophageal cancer was also discussed.

Materials and methods

Six hundred individuals with primary oesophageal carcinoma underwent oesophageal resection in the Department of Surgery II, Kyushu University from 1965 to April 1993. Microscopic sections of the whole resected oesophagus were made from step-sectioned blocks 0.5 cm © 1996 W.B. SaundersCompany Limited

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evaluate both the clinico-pathological characteristics and biological behaviour of each histological pattern. Although cases with minute foci of glandular differentiation in the squamous cell carcinoma sometimes existed, such cases were excluded from the mixed histological types because such small loci of variant histological types would not normally influence the clinico-pathological features, such as metastasis. Using these criteria, seven cases were categorized as mixed histological types of oesophageal cancer, and their histological features and clinico-pathological characteristics were analysed. Immunohistochemical staining for the detection of the p53 protein were used in four particular cases, excluding three with pre-operative irradiation therapy. The polyclonal antibody CM-1 (Novocastra Laborations, Ltd., UK), a rabbit antiserum against the wild-type human p53 protein, was used. For p53 immunostaining, the streptavidin-biotinperoxidase complex method was used. After dewaxing, inactivating the endogenous peroxidase activity, and blocking the cross-reactivity with pre-immune serum (Histogine SAB-PO Kit, Nichirei Ltd., Tokyo, Japan), the sections were incubated overnight at room temperature with the primary antibody (diluted I:100). Localization of the primary antibodies was achieved by a subsequent incubation of a biotinylated anti-primary antibody, and an avidin-biotin complex conjugated with horseradish peroxidase (Histofine SAB-PO Kit). Following these treatments, visualization of the peroxides was made feasible using the diamo benzidine. Counter-staining was done with methylgreen. Each section was then examined with a transmission light microscope and the findings were compared with those for haematoxylin and eosin (H&E) stained sections. The stain reactions were then evaluated as being either positive or negative, and the positive reaction was considered to be present only when strong brown deposits were visible.

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Fig. I. (a) A low-powered view of a protruding type tumour in Case 6. The tumour invasion was restricted to the submucosal layer (H&E, x 2.8). (b) The area of squamous cell carcinoma in the tumour in Case 6 was located mainly in the lateral parts of the tumour (H&E, × 100). (c) The sarcomatous portion of the tumour in Case 6 was located in the centre of the tumour. Spindle-shaped cells were arranged in a striform growth pattern (H&E, x 140). in width and were stained with haematoxylin and eosin (H&E). To identify any mixed histological types of oesophageal cancer in this study, we employed the following criteria: (1) cancer composed of definitely different histological types, (2) the types should both present definite malignant histological features, (3) both be located within the same tumour tissue specimen, and (4) that more than one-third of the area showed a different histological pattern to that of the predominant histology. We applied the last criterion to

Results Using our criteria, seven cases were considered to be oesophageal cancer composed of mixed histological types. In these cases, in addition to ordinary squamous cell carcinoma, a mixture of adenocarcinoma was evident in one, adenoid cystic carcinoma was seen in three, a sarcomatous component was found in two, and basaloid carcinoma and sarcomatous component were each observed in one case. The clinical features of the mixed histological types of oesophageal cancer are shown in Table 1. Age and sex of the patient, and location or size of the lesion, in this type of tumour failed to reveal any significant characteristics. The histopathological characteristics of these tumours are shown in Table 2. All but one of the tumours were elevated lesions (four polypoid lesions and two protruding lesions), and the predominant components of tumour elevation were not squamous cell carcinoma but other variant histological types. Except for one case with tumour invasion of the adventitia, tumours were restricted to the submucosal layer. Thus, in this type of tumour, there was a tendency of protruding growth rather than deepl~¢ invasive growth.

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Fig. 2 (a) A low-powered view of a polypoid type tumour in Case 7. The invasion of the tumour was restricted to the submucosal layer (H&E, x 2.8). (b) The area of squamous cell carcinoma in the lateral part of the tumour in Case 7. Lymphatic vessel permeation ofsquamous cell carcinoma was noted (H&E, x 34). (c) The area of basaloid carcinoma located in the stalk of the polypoid tumour in Case 7 (H&E, x 100). (d) The area of a sarcomatous pattern located in the main part of the polypoid lesion in Case 7 (H&E, x 106). (e) Immunohistochemistry for the detection of the p53 protein in the area of squamous cell carcinoma in Case 7. Immunoreactivity was located in the nuclei of the cancerous g ~O #~", ~ ¢ . . ~ " •~ * ~ : • -. . ". .- .~. ~~, , , - .' .*, ~ ' ~ , ~. • lesion• Note the negative staining in the non-cancerous epithelium on the left side ( x 92). (f) p53 immunostaining in .-:. ~v, ~ -,:. • ... . o ~. .. . ;'~'-~":.!'~"~-~" ' ~ . . . . ~,. " ' "~ - " , the area of a basaloidcarcino matous pattern in Case 7. Positive staining in the nuclei of malignant cells was noted ( x 94). (g) Immunohistochemistry for p53 in the sarcomatous area in Case 7. The nuclei of the tumour cells were also positively stained, although the positivity was somewhat weak compared with other areas of the tumour ( x 230).

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Table 1. Clinical features of the mixed histological types of oesophageal cancer Case

Age/sex

Location of the lesion

Length of the lesion (cm)

Operation

Histology

1

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Middle

8.5 3.0

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5

43/M

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6

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Lower

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Middle

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Adenocarcinoma poorly diff. SCC Adenoid cystic ca. mod. diff. SCC Adenoid cystic ca. poorly diff. SCC Adenoid cystic ca. mod. diff. SCC Sarcomatous component mod. diff. SCC Sarcomatous component poorly diff. SCC Basaloid ca. sarcomatous component mod. diff. SCC

2

65/M

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3

57/M

4

well diff. SCC: well-differentiated squamous cell carcinoma. mod. diff. SCC: moderately differentiated squamous cell carcinoma. poorly diff. SCC: poorly differentiated squamous cell carcinoma. Adenoid cystic ca.: adenoid cystic carcinoma.

Lymphatic vessel permeation by adenocarcinoma and squamous cell carcinoma was seen in one and two cases, respectively, and vascular permeation by adenoid cystic carcinoma was evident in one case. Lymph node metastasis from squamous cell carcinoma and lung metastasis from adenocarcinoma were seen in the cases with lymphatic vessel permeation by adenocarcinoma and squamous cell carcinoma, respectively. Recurrence in the lung and mediastinal lymph nodes was seen in three cases and one case, respectively; two patients also died due to disease recurrence. Figure l(a) shows a low-powered view of a protruding type tumour from case 6. Tumour invasion was restricted to the submucosal layer. The tumour was composed of both a squamous cell carcinoma (Fig. l(b)) and a sarcomatous component (Fig. l(c)), and the elevation of the lesion was mainly composed of the latter component. Figure 2(a) shows a low-powered view of the polypoid type tumour of case 7, and tumour invasion was restricted to the submucosa. In case 7, the lesion was composed ofsquamous cell carcinoma located in the lateral areas of the tumour (Fig. 2(b)), basaloid carcinomatous pattern in the stalk of the polypoid tumour (Fig. 2(c)), and a sarcomatous component in the main part of the polypoid mass (Fig. 2(d)). An immunohistochemical study to detect p53 protein was performed and, in case 7, positive staining of the nucleus of the tumour cells was demonstrated in the areas with squamous cell carcinoma (Fig. 2(e)), basaloid carcinoma (Fig. 2(f)), and the sarcomatous component (Fig. 2(g)). These areas of positive staining were specific in the nuclei of the malignant cells. Table 3 summarizes the data from four cases of an immunohistochemical study for the detection of p53 protein. Positive staining of the nuclei of the tumour cells can be seen in two cases, while, in such cases, both the areas of squamous cell carcinoma and other histological variants were positively stained.

Discussion

The most common tumour in the oesophagus is squamous cell carcinoma, while other histological types of oesophageal tumour only rarely occur. In 1973, Turnbull et al. reviewed 1918 patients with cancer of the oesophagus and demonstrated that there were 66 patients with malignant tumours other than typical epidermoid carcinoma, including 45 with primary adenocarcinoma, 10 with variants of epidermoid carcinoma characterized histologically by prominent glandular or spindle cell features, two malignant melanoma, one mucoepidermoid carcinoma and one oat cell carcinoma.t-" In Japan, Suzuki and Nagayo summarized the data from a nationwide collection of oesophageal tumours other than squamous cell carcinoma, and demonstrated that, in such rare tumours, adenocarcinoma was relatively common followed by undifferentiated carcinoma, adenoacanthoma, non-epithelial malignant tumours, carcinosarcoma, pseudosarcoma, mucoepidermoid carcinoma, adenoid cystic carcinoma, basal cell carcinoma, oat cell carcinoma and carcinoid, among the surgically resected cases. I~ Such histological variations sometimes coexist with ordinary squamous cell carcinoma. In the series of Turnbull et al., j2 10 cases of epidermoid carcinoma with glandular features or with spindle cell metaplasia were identified. Epstein et al. reviewed the literature concerning adenoid cystic carcinoma of the oesophagus and demonstrated that the condition sometimes coexisted with either dysplasia or carcinoma. 3 We also revealed that almost all cases with glandular or mucussecreting components in oesophageal cancer coexist with squamous cell carcinoma. ~'2 However, to our knowledge, there have been no previous reports on cancer of mixed histological types within the oesophagus, although Dodge previously demonstrated seven cases with various blends of squamous cells, glandular, mucoepidermoid and anaplastic structures in the gastro-oesophageal junctionJ 4 In the

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Table 3. Immunohistochemistry for the detection of the p53 protein in mixed histological types of oesophageal cancer Case

Histology

1

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5 6 7

Positive staining (-) (-) (- ) (-) (+ ) (+ ) (+) (+) (+ )

For abbreviations, see Table 1.

current study, we reviewed the cases of oesophageal cancer composed of mixed histological types, in which both components occupied considerable areas in the tumour tissue, in order to evaluate the biological nature and histogenesis of each component. Based on our findings, some pathological characteristics in the cancer of mixed histological types were demonstrated, although no specific features or clinical factors were found. Almost all the tumours were elevated lesions and the less invasive and the predominant components of tumour elevation were not squamous cell carcinoma but other histological variants. Malignant polypoid lesions are occasionally encountered and the most common malignant polyps usually consist of carcinosarcoma or pseudosarcoma. 4'~5 Other rare types of sarcoma, such as leiomyosarcoma, t6synovial sarcoma, 17liposarcoma,~S Triton tumours ~9also showed polypoid growth on most occasions, while primary adenocarcinoma also sometimes showed polypoid growth. 2° Therefore, the elevated growth in most cases in the current study was influenced by the nature of such variations in the histological types. Vessel permeation, patterns of metastasis and recurrence showed no specific tendency. In an attempt to reconstruct the histogenesis of oesophageal cancer, we noted the high incidence ofglandular or mucus-secreting components in oesophageal squamous cell carcinoma, t the frequent coexistence of intraepithelial carcinoma contiguous to the main squamous cell carcinoma in early oesophageal cancer, 2~the close relationship between multiplicity and the existence of intraepithelial carcinoma with squamous cell carcinoma,22 and the high incidence in the coexistence of intraepithelial carcinoma and glandular differentiation, particularly in early cancers.: These results thus support the concept of either a multicentric or epithelial field carcinogenesis of oesophageal cancer. Recently, Wang et al. demonstrated the accumulation of p53 protein not only in the carcinoma but also in the pre-cancerous lesions of the oesophagus by immunohistochemical staining.:3 We also performed an immunohistochemical study for four cases of mixed histological type oesophageal cancer. The results showed that the components of histological variation were all positive in the two positive cases and all were negative in the two negative cases. It is difficult to clarify the process of the histogenesis of each element of mixed histological types. Two hypotheses of the process of such coexistence of histologically different type malignant

tumours in the same cancer tissue exist. One concerns field carcinogenesis, which is the concept that different types of cells are transformed to malignant cells simultaneously. The other hypothesis posits that one type of cancer showed metaplastic change to the other type of cancer. Although whether or not the field carcinogenesis or metaplastic change is the main sequence of the histogenesis of these tumours with mixed histological types still remains unclear, at least the process of genetic change and carcinogenesis would be similar within one case even if the histological features were different. Further investigation using the microdissection method and mutation analysis of p5324 in such tumours of mixed histological types may also provide significant information about carcinogenesis in these tumours.

Acknowledgements The authors would like to thank Mr Brian T. Quinn for the critical reading of this manuscript. This work was supported in part by Grant No. 3986 from the Ministry of Education, Science and Culture, Japan and by a Grant from the Fukuoka Cancer Society.

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Accepted for publication 12 January 1996