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Salivary duct carcinoma: clinicopathological and immunohistochemical studies
Journal of Cranio-Maxillofitcial Surgery (1997) 25, 328-334 © 1997 European Association for Cranio-MaxillofacialSurgery
Salivary duct carcinoma: clinicopathological and immunohistochemical studies E. Martinez-Barba 1, J. A. Cortes-Guardiola 2, A. Minguela-Puras 3, A. Torroba-Caron 1, S. Mendez-Trujillo 4, J. Bermejo-Lopez5
1Pathology Service, 2Oral and Maxillofacial Service, 3Immunology Section, 4Chief of Department, Oral and Maxillofacial Service, sChief of Department, Pathology Service, Virgende la Arrixaca UniversityHospital, 30120 El Pahnar, Murcia, Spain S U M M A R Y . Nine cases of salivary duct carcinoma were reviewed clinically, histologically and immunohistochemically, with special evaluation of biomarkers with prognostic significance (p53, Ki67, c-erbB-2 and DNA content). Eight tumours occurred in the parotid gland and one in the submandibular gland. The average age of the patients (8 males and 1 female) was 62.8 years (range = 47-74 years). Tumour size ranged from 1 to 6 cm (mean = 3.46 cm). Recurrences were found in 33.3% (3 patients), regional metastases in 44.4% (4 patients) and systemic metastases in 33.3% (3 patients). Three patients died of their disease (median survival = 12.3 months), one is alive with the disease (follow-up of 222 months) and 5 are alive without evidence of disease (mean follow-up of 75 months), p53 protein nuclear immunostaining was positive in 66.6% and c-erbB-2 overexpression was observed in 100% of the tumours. Ki 67 positivity ranged from 6.75% to 47.5% of turnout cells (mean = 21.3%). DNA aneuploidy was found in 4 tumours (44.4%) and DNA diploidy in 5 (55.5%). Our results seem to indicate that Ki67 immunostaining can be useful in the evaluation of the biological behaviour of these tumours, as well as the presence of a high proliferative index of aneuploid cells and the presence of distant metastases.
INTRODUCTION
cases of salivary duct carcinoma were identified. Clinical and follow-up data were obtained by reviewing patient records or by personal contact with the patient. Minor salivary glands were not examined. In all cases, the surgical specimens were fixed in 10% formalin, embedded in paraffin, and stained with haematoxylin-eosin, periodic acid-Schiff with and without diastase digestion and alcian blue at pH = 2.6. Additional slides of paraffin blocks were stained by the standard immunohistochemical avidinbiotin peroxidase method in all cases, using commercially available antibodies and appropriate control. For c-erbB-2 oncoprotein overexpression, only membrane staining was considered positive. Its degree of expression was evaluated as negative (-), scattered cells with detectable immunostaining (+ to + +), and strongly positive (+ + +) when distinct membrane staining in most cells was observed, with appropriate controls. Ki67 and p53 staining was evaluated separately by two different pathologists counting 400 consecutive cells per tumour in a premarked area on each slide with a x 100 magnification. All labelled nuclei, regardless of staining intensity, were considered positive and evaluated as (+) (< 5% of neoplastic cells), (+ +) (between 5% and 20%), and (+ + +) (> 20%) and appropriate controls were performed. The rest of the immunoperoxidase stainings were assessed semi-quantitatively, negative as 0, +/- as equivocal and +, + +, and + + + indicating different intensities when definitely positive (Table 1).
Salivary duct carcinoma is a distinctive and aggressive neoplasm of the salivary glands first described by Kleinsasser et al (1968) and integrated in the recent WHO classification (Seifert, 1991). This neoplasm is characterized histologically by a striking resemblance to mammary duct carcinoma and occurs most often in the parotid of middle-aged and older males. Previous reports have not found any association between DNA content and the biological aggressiveness of this type of tumour (Grenko et al., 1995; Felix et al., 1996; Lewis et al., 1996). In contrast, c-erbB-2 overexpression has been described as an independent prognostic parameter associated with aggressive biological behaviour in these patients (Felix et al., 1996). In this study, we describe the clinicopathological and immunohistochemical features of nine cases of salivary duct carcinoma with special emphasis on the evaluation of biomarkers with prognostic significance, correlating the results with the biological behaviour of the tumours and patient survival.
PATIENTS AND METHODS The pathology files of Virgen de la Arrixaca Hospital were examined for all cases of major salivary gland cancer from 1968 to 1996 (136 cases). Tissue slides from all of these neoplasms were reviewed and nine 328
Salivary duct carcinoma 329
Table 1 - Immunohistochemical and cytometrical findings in nine cases of salivary duct carcinoma Antibody
1
*Pan-Cytokeratin +++ *CAM 5.2 +++ *EMA +++ *pCEA +++ *S- 100 protein 0 *MSA 0 *Oestrogen Receptor 0 (Clon 1D-5) **p53 protein (DO-7) + **Ki67 antigen ++ ***c-erbB-2 D+++ Ploidy diploid PId/PIa 7.32
2
3
4
+++ +++ +++ +++ 0 0 0
+++ +++ +++ +++ 0 0 0
+++ +++ +++ +++ 0 0 0
+++ ++ D+++ diploid 24.9
+ +++ D+++ aneuploid 3.7/27
+ ++ D+++ diploid 2.5
Case number 5
6
7
8
9
+++ +++ +++ +++ 0 0 0
+++ +++ +++ + 0 0 0
+++ +++ +++ +++ 0 0 0
+++ +++ +++ +++ 0 0 0
+++ +++ +++ +++ 0 0 0
+ +++ D+++ aneuploid 6.8/23
+ ++ F+ aneuploid 16.1/8.8
++ +++ D+++ diploid 4.8
+ ++ D+++ diploid 8.6
+ +++ D+++ aneuploid 5.3/24
EMA: epithelial membrane antigen; pCEA: polyclonal carcinoembryonic antigen; MSA: muscle-specificactin; PId: proliferative index of diploid population; PIa: proliferative index of aneudiploid population. *Staining was evaluated as: 0 = negative; +/- = equivocal, +, ++ and +++ = increasing intensity of positive staining. **Staining was evaluated as: (+) (< 5% of neoplastic cells), (++) (between 5% and 20%), and (+++) (> 20%); D " diffuse, F = focal. ***Staining was evaluated as: negative (-), scattered cells with detectable immunostaining (+ to + +) and strongly positive (+ + +); D = diffuse, F = focal. Source and dilution: Biogenex (San Ramon, CA) (Pan-Cytokeratin 1:50), Becton Dickinson (San Jose, CA) (CAM 5.2 Prediluted), Biomeda (Foster City, CA) (EMA Prediluted), DAKO (Denmark) (CEA 1:400, S-100 1:300, MSA 1:50, Oestrogen Receptor 1:30, p53 1:25, Ki67 1:50, c-erbB-2 1:100).
Flow cytometry
RESULTS
Nuclear suspensions in all cases were prepared from 50 g m paraffin embedded tissue sections using the m e t h o d described by Hedleyet al (1983). For cell cycle analysis, a single stain procedure with propidium iodine was developed using CellCycle-kit (Becton Dickinson, San Jos6, CA). Samples were filtered by 53 g m mesh and measured on a F A C S o r t flow cytometer using the CellQuest software (BD) and counting at least 15 000 cells/sample. Nuclei from a deparaffinized n o r m a l - l y m p h node was previously examined as external standard. D N A histograms were analysed by M o d F i t software (BD), defining aneuploid samples as those histograms having a second peak that did not fall in the expected channels o f the G1 or G2 + M peaks. The proliferative index was regarded as the percentage o f cells within the S and G2 + M phases.
Clinicopathological and histological findings
Statistical analysis For statistical purposes, patients were classified into two groups: died o f disease, and alive with or without evidence o f disease. Categorical variables analysis (sex, patient status, ploidy, perineural invasion, l y m p h n o d e metastasis and distant metastasis) was performed using the Fisher's exact test. The Student t-test was used to analyse quantitative variables: age, t u m o u r size, p53, Ki67, c-erbB2, proliferative index o f diploid cells and proliferative index o f aneuploid population. Quantitative variable relationship was m a d e by correlation analysis. Only P values below 0.05 calculated by the B M D P software were considered significant.
Clinicopathological findings are summarized in Table 2. The patient population comprised 8 males and 1 female, who ranged in age f r o m 47 to 74 years, with a m e a n o f 62.8 years. T u m o u r s usually appeared clinically as painless nodules in the parotid gland, and were accompanied by facial nerve paralysis in two cases. One t u m o u r was found in the submandibular gland. One patient had a painful nodule and history o f a pleomorphic a d e n o m a 20 years before. All patients were treated surgically with total to radical excision; radical neck dissection was performed in 6 patients. Eight patients received postoperative radiotherapy and one patient received postoperative chemotherapy. Local recurrences were f o u n d in 3 patients, regional metastases in 4 patients and systemic metastases in 3 patients. Three patients died as a result o f their tumour. Five patients have not shown local recurrences and remain disease-free after 3 months, 2 years, 5 years, 11 years and 13 years, respectively. One patient with 6 local recurrences remains alive with disease 185 years after diagnosis. T u m o u r s ranged in size from 1 to 6 cm (mean = 3.46 cm). The most c o m m o n appearance was a poorly circumscribed, multi-nodular, solid, grey-white mass, although two t u m o u r s measuring 3 and 5.5 cm respectively were well circumscribed but not encapsulated. Microscopically, all turnouts contained areas o f well-differentiated ductal structures (Fig. 1) resembling m a m m a r y ductal carcinoma in situ, showing central comedonecrosis associated with a cribriform,
330 Journalof Cranio-Maxillofacial Surgery Table 2 Clinical findings in nine patients with salivary duct carcinoma Case No.
Sex/Age (yrs)
Clinical presentation
Site/Size of tumour (cm)
Treatment
Follow-up
Painless nodule; facial nerve paralysis Unknown Painless nodule
Right parotid/1
RP, RND and PR
NED at 13 yrs
Submandibular/2.3 Left parotid/5.5
Excision and PR RP and PR
NED at 11 yrs Metastases in bone at 8 too; DOD at 12 mo Metastases in neck lymph nodes at time of diagnosis. NED at 5 yrs Local recurrence at 4 mo; cervical lymph node metastases at 8 mo; metastases in lung at 10 too; DOD at 14 mo Cervical lymph node metastases at 3 mo; local recurrence at 5 yr, 6½Yr, 10yr, 12yr, 16yr, and 161 yr. AWD at 18½yr. NED at 3 mo NED at 2 yrs Local recurrence and cervical lymph node metastases at 5 mo. Metastases in lung at 8 mo. DOD at 11 mo.
1
Ml68
2 3
M/58 M/66
4
M/63
Painless nodule; facial nerve paralysis
Left parotid/1.5
RE RND and PR
5
M/47
Painful nodule; history of pleomorphic adenoma 20 years previously
Left parotid/3
RE RND, PR and P Chemo
6
M/54
Painless nodule
Left parotid/5
RE RND and PR
7 8 9
Ml69 F/67 Ml74
Painless nodule Painless nodule Painless nodule
Right parotid/5.5 Left parotid/1.4 Right parotid/6
TP and PR RR RND and PR TP and RND
RP: radical parotidectomy; TP: total parotidectomy; RND: radical neck dissection; PR: postoperative radiotherapy; P Chemo: postoperative chemotherapy; NED: no evidence of disease; DOD: died of disease, AWD: Alive with disease.
F i g . 1 - Areas of well-differentiated ductal structures showing central comedonecrosis (haematoxylin-eosin stain, original magnification x 100).
Fig. 2 - Infiltrating component is associated with dense fibrous tissue (haematoxylin-eosin stain, original magnification x 100).
solid, p a p i l l a r y or d e s m o p l a s t i c architecture (Fig. 2). M o s t o f the cells h a d e o s i n o p h i l i c c y t o p l a s m , usually showing a s h a r p l y defined cell margin. Nuclei were single a n d often vesicular with coarsely g r a n u l a r chrom a t i n (Fig. 3). Some nuclei c o n t a i n a single nucleoli, sometimes p r o m i n e n t . Cytologic pleomorphism v a r i e d f r o m slight to m o d e r a t e , even within different areas o f the same t u m o u r . M i t o t i c figures v a r i e d f r o m 1 to 16 p e r 10 h i g h - p o w e r fields. Small quantities o f l u m i n a l m u c i n were n o t e d in all cases. However, no i n t r a c e l l u l a r m u c i n was n o t e d . F i b r o s i s was p r o m i nent in three tumours. R u p t u r e o f d u c t a l e p i t h e l i u m with collections o f f o a m y histiocytes was o b s e r v e d in case 7 (Fig. 4).
P e r i n e u r a l invasion was n o t e d in six t u m o u r s (cases 1, 2, 3, 4, 5 a n d 9). L y m p h n o d e m e t a s t a s e s were f o u n d in 4 patients; t h e y r e t a i n e d identical features to their p r i m a r y t u m o u r s . I n f i l t r a t i o n o f vascul a r a n d l y m p h a t i c structures was p r o m i n e n t in case 5. Cases 1, 7 a n d 8 h a d ducts w i t h only p a r t i a l involvem e n t b y the n e o p l a s t i c cells with m i c r o p a p i l l a r y intraluminal projection and pseudocribriform cellular p r o l i f e r a t i o n c r e a t i n g a p a t t e r n o f so-called ' R o m a n b r i d g e s ' , w i t h o u t central c o m e d o n e c r o s i s (Fig. 5). However, lesions t h a t r e p r e s e n t e d a n i n t r a d u c t a l c o m p o n e n t were rare (< 5% o f the t u m o u r mass). In one t u m o u r , i n t r a d u c t a l , n o n - b i r e f r i n g e n t , n e e d l e - s h a p e d c r y s t a l l o i d s were found.
Salivaryduct carcinoma 331
Fig.
3 - The tumour is composedof cellswith vesicularnuclei, singlenucleoliand eosinophiliccytoplasm(haematoxylin-eosin stain, originalmagnificationx 400).
Rupture of ductal epitheliumwith collectionsof foamy histiocytes(haematoxylin-eosinstain, originalmagnification x 100).
Fig. 5 - Note the intraductalproliferationwith so-called'Roman bridges' (haematoxylin-eosinstain, originalmagnificationx 200).
Fig. 6 -
Statistical analysis did not find any association between sex, age, perineural invasion or lymph node metastasis with the clinical outcome in these patients, and also no significant differences between tumour size and patient outcome was found. In contrast, an association seems to exist between the clinical outcome and the presence of distant metastasis (P < 0.012).
(mean = 11.3%). C-erbB-2 immunoreactivity was focal and weakly positive (+) in 1 tumour and strong and diffuse (+++) in eight tumours (Fig. 7). p53 and c-erbB-2 positivity did not show any significant differences between the two groups of patients, whereas Ki67 positivity was significantly higher (> 20% of the tumour cells) in patients who died of the disease (P < 0.01).
Immunohistochemistry
Flow cytometry
The immunohistochemical findings are summarized in Table 1. Staining for cytokeratins (low and high molecular weight) and epithelial membrane antigen were positive in all 9 cases. Polyclonal carcinoembryonic antigen was strongly positive in eight tumours (+++) and weak and focal in case 6. Stains for oestrogen receptors were negative in all tumours. The negativity for muscle-specific actin and S-100 protein in all 9 cases showed that there was no evidence of myoepithelial differentiation. Ki67 nuclear positivity (Fig. 6) ranged from 6.75% to 47.5% (mean = 21.3%) and p53 from 0% to 72.5%
Four tumours displayed DNA aneuploidy (hyperdiploid) and 5 were DNA diploid. It is important to mention that all the 5 patients showing diploid tumours are alive without evidence of disease. However, 3 out of 4 patients with aneuploid tumours showed distant metastases and died of the disease 11, 12 and 14 months after diagnosis, respectively. The fourth patient bearing an aneuploid tumour (case 6) suffered from six local recurrences without distant metastasis, being alive with disease 18~ years after diagnosis. Among patients bearing aneuploid tumours, those who died of their disease showed a
Fig. 4 -
Ki67 immunostainingshowingnuclearstainingin some tumour cells (originalmagnificationx 200).
332 Journalof Cranio-MaxillofacialSurgery
C-erbB-2immunostaining showing intense membrane staining (original magnification x 400).
Fig. 7 -
tumoral cell population with a high proliferative index (23%, 24% and 27%, respectively), whereas case 6 showed a tumoral cell population with a low proliferative index (8.8%). The flow cytometric results are detailed in Table 1. Interestingly, statistical analysis showed that patients dying of their disease (P < 0.0005) and with distant metastasis (P < 0.0005) seemed to show aneuploid cells with a proliferative index significantly higher than patients alive with or without evidence of disease. In addition, positive correlation between Ki67 positivity and the proliferative index of aneuploid cells was found (r = 0.736, P < 0.05).
DISCUSSION Salivary duct carcinoma is a distinctive and aggressive neoplasm, characterized histologically by a striking resemblance to ductal carcinoma of the breast, that occurs predominantly in the major salivary glands of middle-aged and older males. The clinical and histological characteristics of our patients are similar to those in other series (Garland et al., 1984; Brandwein et al., 1990; Simpson et al., 1991; Colmenero et al., 1993; Delgado et al., 1993; Grenko et al., 1995; Lewis et al., 1996). Six of our patients had a history, of 18 months or less, of a painless mass. However, one patient had a parotid swelling for 12 years which enlarged rapidly over 7 months. Case 5 had a history of pleomorphic adenoma 20 years before, it was treated by surgery and radiotherapy. However, there was no histological evidence of a pre-existing benign mixed tumour. The histological appearance in our cases resembled ductal carcinoma of the breast, particularly the comedo-like pattern of necrosis which we found in all our cases. An intraductal component was identified in 3 tumours, although these lesions represented less than 5% of the tumour mass. Local recurrence in salivary duct carcinoma has been reported in about 45% of the cases (Brandwein et al., 1990). The incidence of local recurrence in our
series is lower. However, one patient has presented six local recurrences during an interval of 18~ years after diagnosis. Luna et al (1987) and Brandwein et al (1990) reported distant metastases in 66% of 30 patients and in 57% of 58 patients, respectively; however, in our study this incidence is lower. The presence of cervical lymph node metastases in this study is in accordance with previous reports, which ranged from 40 to 72% (Garland et al., 1984; Brandwein et al., 1990). Perineural invasion has been frequently observed (Delgado et al., 1993; Lewis et al., 1996). In our study, perineural invasion was found in 6 patients. Of our 3 patients without perineural invasion, one is alive with disease and the others are without evidence of disease 222, 24 and 3 months after surgery, respectively. Although salivary duct carcinoma was recognized as an aggressive neoplasm, in a review of previous studies (Hui et al., 1986; Afzelius et al., 1987; Brandwein et al., 1990; Simpson et al., 1991; Delgado et al., 1993; McKinney et al., 1994) a significant minority (approximately 25-30%) of patients are well with prolonged disease-free survival. However, in our short series, 6 patients are alive with or without disease. We have not found any clinicopathological feature of salivary duct carcinoma that could discriminate between those patients alive with or without disease and those dying of disease, except the presence of distant metastases. Our study shows that salivary duct carcinoma is not uncommon among malignant tumours of salivary glands, constituting up to 15% at this site. Grenko et al (1995) found 12 salivary duct carcinomas among 145 primary parotid tumours, and, in a review of 4068 salivary glands tumours, Seifert and Caselitz (1989) reported 37 cases of salivary duct carcinoma (0.9%). Taking these results into account, it is possible to suppose that this tumour could be under-recognized by those pathologists unfamiliar with it, and that regional differences in the tumour prevalence could exist. Our immunohistochemical findings, with cytokeratins (low and high molecular weight), epithelial membrane antigen and carcinoembryonic antigen positivity in tumour cells are in accordance with previous reports (Brandwein et al., 1990; Simpson et al., 1991; Delgado et al., 1993; Lewis et al., 1996). As previously described (Delgado et al., 1993; Lewis et al., 1996), our cases showed a consistent absence of S-100 protein and actin muscle-specific positivity showing that there was no evidence of myoepithelial differentiation. In addition, oestrogen receptor immunostaining was negative according to others studies (McKinney et al., 1994; Hellquist et al., 1994). Ockner et al (1994) suggested that the presence or absence of oestrogen receptor could be a distinguishing feature between salivary duct carcinoma and metastatic breast carcinoma, a hypothesis which is in agreement with our results.
Salivary duct carcinoma
In humans, several studies indicate that overexpression of c-erbB-2 oncoprotein is associated with a poor prognosis in some malignant tumours (Ro et al., 1989; Soomro et al., 1991). In salivary gland tumours, several studies have evaluated c-erbB-2 expression (Kernohan et al., 1991; Sugano et al., 1992; Barnes et al., 1994; Hellquist et al., 1994; Felix et al., 1996). Sugano et al (1992) suggested that salivary gland carcinomas showing c-erbB-2 protein positivity were aggressive in nature and had a poor prognosis, therefore these patients might benefit from a more aggressive therapy. Felix et al (1996) found that c-erbB-2 overexpression was a significant survival predictor. In others reports, c-erbB-2 overexpression ranged from 25 to 88% (Hellquist et al., 1994; Barnes et al., 1994), whereas in our study c-erbB-2 positivity did not show any significant differences between patients alive or dead from the disease. In salivary gland tumours, several studies (Soini et al., 1992; Deguchi et al., 1993) have assessed p53 immunostaining and aggressive clinical outcome. Soini et al (1992) also found correlation between DNA aneuploidy and p53 immunostaining in salivary gland tumours. In contrast, other reports (Hellquist et al., 1994; Felix et al., 1996), together with our results, have failed to find any association between p53 positivity and patient outcome. The Ki67 immunostaining reacts with a nuclear antigen present in proliferating human cells. A study using Ki67 immunostaining found high proliferative activity in this type of tumour, associated with poor patient survival (Hellquist et al., 1994). In our study the Ki67 positivity was significantly higher (> 20% of tumour cells) in patients dying of the disease. These results seem to indicate that Ki67 immunostaining can be useful in the evaluation of the biological aggressiveness of this type of tumour. DNA content in salivary gland tumours has been assessed in several studies, with differing results. Two reports describing 31 and 24 salivary duct carcinomas (Lewis et al., 1996; Felix et al., 1996) did not find any correlation between DNA ploidy or S-phase values and clinical outcome. In contrast, others studies of malignant salivary gland tumours have found correlation between DNA aneuploidy or phase fraction with the histological grade and prognosis (Hamper et al., 1989; Barnes et al., 1994). In our study, we have found a positive correlation between tumours with high proliferative index of aneuploid cells and the presence of distant metastases, as well as with fatal clinical outcome of patients. Interestingly, case 6, who is alive 222 months after diagnosis, showed an aneuploid tumour with a low proliferative index of aneuploid cells and a moderate Ki67 positivity, facts which could explain this patient's prolonged survival. In conclusion, salivary duct carcinoma may not be as rare as previously considered. Although this
333
tumour was recognized as an aggressive neoplasm, in our study six patients are alive with or without disease after 3 months, 2 years, 5 years, 11 years, 13 years and 18~ years, respectively. We think that the most effective treatment is the use of aggressive therapy (radical surgery, radical neck dissection and postoperative radiotherapy), although the results are unequal. In addition, our results seem to indicate that Ki67 immunostaining can be useful in the evaluation of the biological behaviour of these tumours, as well as the presence of a high proliferative index of aneuploid cells and the presence of distant metastases. Nonetheless, further research, with more extensive series, is needed to confirm our findings. Acknowledgements The authors thank Prof. Dr Manuel Canteras Jordana for his assistance with statistical analysis and Ms M" Dolores Lapuente, Ms Dolores Ortufio, Ms M ~ Luisa Fern/mdez, Mr Francisco C/movas and Mr Mateo Fern/mdez for their excellent technical assistance.
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duct adenocarcinoma. Histopathology 18 (1991) 229-235 Soini, E, D. Kamel, K. Nuorva, et al." Low p53 protein expression
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Dr Enrique Martinez-Barba Pathology Service Virgen De La Arrixaca University Hospital 30120 E1Palmar Murcia Spain
Paper received 16 June 1997 Accepted 15 Oct. 1997
Salivary duct carcinoma: clinicopathological and immunohistochemical studies E. Martinez-Barba 1, J. A. Cortes-Guardiola 2, A. Minguela-Puras 3, A. Torroba-Caron 1, S. Mendez-Trujillo 4, J. Bermejo-Lopez5
1Pathology Service, 2Oral and Maxillofacial Service, 3Immunology Section, 4Chief of Department, Oral and Maxillofacial Service, sChief of Department, Pathology Service, Virgende la Arrixaca UniversityHospital, 30120 El Pahnar, Murcia, Spain S U M M A R Y . Nine cases of salivary duct carcinoma were reviewed clinically, histologically and immunohistochemically, with special evaluation of biomarkers with prognostic significance (p53, Ki67, c-erbB-2 and DNA content). Eight tumours occurred in the parotid gland and one in the submandibular gland. The average age of the patients (8 males and 1 female) was 62.8 years (range = 47-74 years). Tumour size ranged from 1 to 6 cm (mean = 3.46 cm). Recurrences were found in 33.3% (3 patients), regional metastases in 44.4% (4 patients) and systemic metastases in 33.3% (3 patients). Three patients died of their disease (median survival = 12.3 months), one is alive with the disease (follow-up of 222 months) and 5 are alive without evidence of disease (mean follow-up of 75 months), p53 protein nuclear immunostaining was positive in 66.6% and c-erbB-2 overexpression was observed in 100% of the tumours. Ki 67 positivity ranged from 6.75% to 47.5% of turnout cells (mean = 21.3%). DNA aneuploidy was found in 4 tumours (44.4%) and DNA diploidy in 5 (55.5%). Our results seem to indicate that Ki67 immunostaining can be useful in the evaluation of the biological behaviour of these tumours, as well as the presence of a high proliferative index of aneuploid cells and the presence of distant metastases.
INTRODUCTION
cases of salivary duct carcinoma were identified. Clinical and follow-up data were obtained by reviewing patient records or by personal contact with the patient. Minor salivary glands were not examined. In all cases, the surgical specimens were fixed in 10% formalin, embedded in paraffin, and stained with haematoxylin-eosin, periodic acid-Schiff with and without diastase digestion and alcian blue at pH = 2.6. Additional slides of paraffin blocks were stained by the standard immunohistochemical avidinbiotin peroxidase method in all cases, using commercially available antibodies and appropriate control. For c-erbB-2 oncoprotein overexpression, only membrane staining was considered positive. Its degree of expression was evaluated as negative (-), scattered cells with detectable immunostaining (+ to + +), and strongly positive (+ + +) when distinct membrane staining in most cells was observed, with appropriate controls. Ki67 and p53 staining was evaluated separately by two different pathologists counting 400 consecutive cells per tumour in a premarked area on each slide with a x 100 magnification. All labelled nuclei, regardless of staining intensity, were considered positive and evaluated as (+) (< 5% of neoplastic cells), (+ +) (between 5% and 20%), and (+ + +) (> 20%) and appropriate controls were performed. The rest of the immunoperoxidase stainings were assessed semi-quantitatively, negative as 0, +/- as equivocal and +, + +, and + + + indicating different intensities when definitely positive (Table 1).
Salivary duct carcinoma is a distinctive and aggressive neoplasm of the salivary glands first described by Kleinsasser et al (1968) and integrated in the recent WHO classification (Seifert, 1991). This neoplasm is characterized histologically by a striking resemblance to mammary duct carcinoma and occurs most often in the parotid of middle-aged and older males. Previous reports have not found any association between DNA content and the biological aggressiveness of this type of tumour (Grenko et al., 1995; Felix et al., 1996; Lewis et al., 1996). In contrast, c-erbB-2 overexpression has been described as an independent prognostic parameter associated with aggressive biological behaviour in these patients (Felix et al., 1996). In this study, we describe the clinicopathological and immunohistochemical features of nine cases of salivary duct carcinoma with special emphasis on the evaluation of biomarkers with prognostic significance, correlating the results with the biological behaviour of the tumours and patient survival.
PATIENTS AND METHODS The pathology files of Virgen de la Arrixaca Hospital were examined for all cases of major salivary gland cancer from 1968 to 1996 (136 cases). Tissue slides from all of these neoplasms were reviewed and nine 328
Salivary duct carcinoma 329
Table 1 - Immunohistochemical and cytometrical findings in nine cases of salivary duct carcinoma Antibody
1
*Pan-Cytokeratin +++ *CAM 5.2 +++ *EMA +++ *pCEA +++ *S- 100 protein 0 *MSA 0 *Oestrogen Receptor 0 (Clon 1D-5) **p53 protein (DO-7) + **Ki67 antigen ++ ***c-erbB-2 D+++ Ploidy diploid PId/PIa 7.32
2
3
4
+++ +++ +++ +++ 0 0 0
+++ +++ +++ +++ 0 0 0
+++ +++ +++ +++ 0 0 0
+++ ++ D+++ diploid 24.9
+ +++ D+++ aneuploid 3.7/27
+ ++ D+++ diploid 2.5
Case number 5
6
7
8
9
+++ +++ +++ +++ 0 0 0
+++ +++ +++ + 0 0 0
+++ +++ +++ +++ 0 0 0
+++ +++ +++ +++ 0 0 0
+++ +++ +++ +++ 0 0 0
+ +++ D+++ aneuploid 6.8/23
+ ++ F+ aneuploid 16.1/8.8
++ +++ D+++ diploid 4.8
+ ++ D+++ diploid 8.6
+ +++ D+++ aneuploid 5.3/24
EMA: epithelial membrane antigen; pCEA: polyclonal carcinoembryonic antigen; MSA: muscle-specificactin; PId: proliferative index of diploid population; PIa: proliferative index of aneudiploid population. *Staining was evaluated as: 0 = negative; +/- = equivocal, +, ++ and +++ = increasing intensity of positive staining. **Staining was evaluated as: (+) (< 5% of neoplastic cells), (++) (between 5% and 20%), and (+++) (> 20%); D " diffuse, F = focal. ***Staining was evaluated as: negative (-), scattered cells with detectable immunostaining (+ to + +) and strongly positive (+ + +); D = diffuse, F = focal. Source and dilution: Biogenex (San Ramon, CA) (Pan-Cytokeratin 1:50), Becton Dickinson (San Jose, CA) (CAM 5.2 Prediluted), Biomeda (Foster City, CA) (EMA Prediluted), DAKO (Denmark) (CEA 1:400, S-100 1:300, MSA 1:50, Oestrogen Receptor 1:30, p53 1:25, Ki67 1:50, c-erbB-2 1:100).
Flow cytometry
RESULTS
Nuclear suspensions in all cases were prepared from 50 g m paraffin embedded tissue sections using the m e t h o d described by Hedleyet al (1983). For cell cycle analysis, a single stain procedure with propidium iodine was developed using CellCycle-kit (Becton Dickinson, San Jos6, CA). Samples were filtered by 53 g m mesh and measured on a F A C S o r t flow cytometer using the CellQuest software (BD) and counting at least 15 000 cells/sample. Nuclei from a deparaffinized n o r m a l - l y m p h node was previously examined as external standard. D N A histograms were analysed by M o d F i t software (BD), defining aneuploid samples as those histograms having a second peak that did not fall in the expected channels o f the G1 or G2 + M peaks. The proliferative index was regarded as the percentage o f cells within the S and G2 + M phases.
Clinicopathological and histological findings
Statistical analysis For statistical purposes, patients were classified into two groups: died o f disease, and alive with or without evidence o f disease. Categorical variables analysis (sex, patient status, ploidy, perineural invasion, l y m p h n o d e metastasis and distant metastasis) was performed using the Fisher's exact test. The Student t-test was used to analyse quantitative variables: age, t u m o u r size, p53, Ki67, c-erbB2, proliferative index o f diploid cells and proliferative index o f aneuploid population. Quantitative variable relationship was m a d e by correlation analysis. Only P values below 0.05 calculated by the B M D P software were considered significant.
Clinicopathological findings are summarized in Table 2. The patient population comprised 8 males and 1 female, who ranged in age f r o m 47 to 74 years, with a m e a n o f 62.8 years. T u m o u r s usually appeared clinically as painless nodules in the parotid gland, and were accompanied by facial nerve paralysis in two cases. One t u m o u r was found in the submandibular gland. One patient had a painful nodule and history o f a pleomorphic a d e n o m a 20 years before. All patients were treated surgically with total to radical excision; radical neck dissection was performed in 6 patients. Eight patients received postoperative radiotherapy and one patient received postoperative chemotherapy. Local recurrences were f o u n d in 3 patients, regional metastases in 4 patients and systemic metastases in 3 patients. Three patients died as a result o f their tumour. Five patients have not shown local recurrences and remain disease-free after 3 months, 2 years, 5 years, 11 years and 13 years, respectively. One patient with 6 local recurrences remains alive with disease 185 years after diagnosis. T u m o u r s ranged in size from 1 to 6 cm (mean = 3.46 cm). The most c o m m o n appearance was a poorly circumscribed, multi-nodular, solid, grey-white mass, although two t u m o u r s measuring 3 and 5.5 cm respectively were well circumscribed but not encapsulated. Microscopically, all turnouts contained areas o f well-differentiated ductal structures (Fig. 1) resembling m a m m a r y ductal carcinoma in situ, showing central comedonecrosis associated with a cribriform,
330 Journalof Cranio-Maxillofacial Surgery Table 2 Clinical findings in nine patients with salivary duct carcinoma Case No.
Sex/Age (yrs)
Clinical presentation
Site/Size of tumour (cm)
Treatment
Follow-up
Painless nodule; facial nerve paralysis Unknown Painless nodule
Right parotid/1
RP, RND and PR
NED at 13 yrs
Submandibular/2.3 Left parotid/5.5
Excision and PR RP and PR
NED at 11 yrs Metastases in bone at 8 too; DOD at 12 mo Metastases in neck lymph nodes at time of diagnosis. NED at 5 yrs Local recurrence at 4 mo; cervical lymph node metastases at 8 mo; metastases in lung at 10 too; DOD at 14 mo Cervical lymph node metastases at 3 mo; local recurrence at 5 yr, 6½Yr, 10yr, 12yr, 16yr, and 161 yr. AWD at 18½yr. NED at 3 mo NED at 2 yrs Local recurrence and cervical lymph node metastases at 5 mo. Metastases in lung at 8 mo. DOD at 11 mo.
1
Ml68
2 3
M/58 M/66
4
M/63
Painless nodule; facial nerve paralysis
Left parotid/1.5
RE RND and PR
5
M/47
Painful nodule; history of pleomorphic adenoma 20 years previously
Left parotid/3
RE RND, PR and P Chemo
6
M/54
Painless nodule
Left parotid/5
RE RND and PR
7 8 9
Ml69 F/67 Ml74
Painless nodule Painless nodule Painless nodule
Right parotid/5.5 Left parotid/1.4 Right parotid/6
TP and PR RR RND and PR TP and RND
RP: radical parotidectomy; TP: total parotidectomy; RND: radical neck dissection; PR: postoperative radiotherapy; P Chemo: postoperative chemotherapy; NED: no evidence of disease; DOD: died of disease, AWD: Alive with disease.
F i g . 1 - Areas of well-differentiated ductal structures showing central comedonecrosis (haematoxylin-eosin stain, original magnification x 100).
Fig. 2 - Infiltrating component is associated with dense fibrous tissue (haematoxylin-eosin stain, original magnification x 100).
solid, p a p i l l a r y or d e s m o p l a s t i c architecture (Fig. 2). M o s t o f the cells h a d e o s i n o p h i l i c c y t o p l a s m , usually showing a s h a r p l y defined cell margin. Nuclei were single a n d often vesicular with coarsely g r a n u l a r chrom a t i n (Fig. 3). Some nuclei c o n t a i n a single nucleoli, sometimes p r o m i n e n t . Cytologic pleomorphism v a r i e d f r o m slight to m o d e r a t e , even within different areas o f the same t u m o u r . M i t o t i c figures v a r i e d f r o m 1 to 16 p e r 10 h i g h - p o w e r fields. Small quantities o f l u m i n a l m u c i n were n o t e d in all cases. However, no i n t r a c e l l u l a r m u c i n was n o t e d . F i b r o s i s was p r o m i nent in three tumours. R u p t u r e o f d u c t a l e p i t h e l i u m with collections o f f o a m y histiocytes was o b s e r v e d in case 7 (Fig. 4).
P e r i n e u r a l invasion was n o t e d in six t u m o u r s (cases 1, 2, 3, 4, 5 a n d 9). L y m p h n o d e m e t a s t a s e s were f o u n d in 4 patients; t h e y r e t a i n e d identical features to their p r i m a r y t u m o u r s . I n f i l t r a t i o n o f vascul a r a n d l y m p h a t i c structures was p r o m i n e n t in case 5. Cases 1, 7 a n d 8 h a d ducts w i t h only p a r t i a l involvem e n t b y the n e o p l a s t i c cells with m i c r o p a p i l l a r y intraluminal projection and pseudocribriform cellular p r o l i f e r a t i o n c r e a t i n g a p a t t e r n o f so-called ' R o m a n b r i d g e s ' , w i t h o u t central c o m e d o n e c r o s i s (Fig. 5). However, lesions t h a t r e p r e s e n t e d a n i n t r a d u c t a l c o m p o n e n t were rare (< 5% o f the t u m o u r mass). In one t u m o u r , i n t r a d u c t a l , n o n - b i r e f r i n g e n t , n e e d l e - s h a p e d c r y s t a l l o i d s were found.
Salivaryduct carcinoma 331
Fig.
3 - The tumour is composedof cellswith vesicularnuclei, singlenucleoliand eosinophiliccytoplasm(haematoxylin-eosin stain, originalmagnificationx 400).
Rupture of ductal epitheliumwith collectionsof foamy histiocytes(haematoxylin-eosinstain, originalmagnification x 100).
Fig. 5 - Note the intraductalproliferationwith so-called'Roman bridges' (haematoxylin-eosinstain, originalmagnificationx 200).
Fig. 6 -
Statistical analysis did not find any association between sex, age, perineural invasion or lymph node metastasis with the clinical outcome in these patients, and also no significant differences between tumour size and patient outcome was found. In contrast, an association seems to exist between the clinical outcome and the presence of distant metastasis (P < 0.012).
(mean = 11.3%). C-erbB-2 immunoreactivity was focal and weakly positive (+) in 1 tumour and strong and diffuse (+++) in eight tumours (Fig. 7). p53 and c-erbB-2 positivity did not show any significant differences between the two groups of patients, whereas Ki67 positivity was significantly higher (> 20% of the tumour cells) in patients who died of the disease (P < 0.01).
Immunohistochemistry
Flow cytometry
The immunohistochemical findings are summarized in Table 1. Staining for cytokeratins (low and high molecular weight) and epithelial membrane antigen were positive in all 9 cases. Polyclonal carcinoembryonic antigen was strongly positive in eight tumours (+++) and weak and focal in case 6. Stains for oestrogen receptors were negative in all tumours. The negativity for muscle-specific actin and S-100 protein in all 9 cases showed that there was no evidence of myoepithelial differentiation. Ki67 nuclear positivity (Fig. 6) ranged from 6.75% to 47.5% (mean = 21.3%) and p53 from 0% to 72.5%
Four tumours displayed DNA aneuploidy (hyperdiploid) and 5 were DNA diploid. It is important to mention that all the 5 patients showing diploid tumours are alive without evidence of disease. However, 3 out of 4 patients with aneuploid tumours showed distant metastases and died of the disease 11, 12 and 14 months after diagnosis, respectively. The fourth patient bearing an aneuploid tumour (case 6) suffered from six local recurrences without distant metastasis, being alive with disease 18~ years after diagnosis. Among patients bearing aneuploid tumours, those who died of their disease showed a
Fig. 4 -
Ki67 immunostainingshowingnuclearstainingin some tumour cells (originalmagnificationx 200).
332 Journalof Cranio-MaxillofacialSurgery
C-erbB-2immunostaining showing intense membrane staining (original magnification x 400).
Fig. 7 -
tumoral cell population with a high proliferative index (23%, 24% and 27%, respectively), whereas case 6 showed a tumoral cell population with a low proliferative index (8.8%). The flow cytometric results are detailed in Table 1. Interestingly, statistical analysis showed that patients dying of their disease (P < 0.0005) and with distant metastasis (P < 0.0005) seemed to show aneuploid cells with a proliferative index significantly higher than patients alive with or without evidence of disease. In addition, positive correlation between Ki67 positivity and the proliferative index of aneuploid cells was found (r = 0.736, P < 0.05).
DISCUSSION Salivary duct carcinoma is a distinctive and aggressive neoplasm, characterized histologically by a striking resemblance to ductal carcinoma of the breast, that occurs predominantly in the major salivary glands of middle-aged and older males. The clinical and histological characteristics of our patients are similar to those in other series (Garland et al., 1984; Brandwein et al., 1990; Simpson et al., 1991; Colmenero et al., 1993; Delgado et al., 1993; Grenko et al., 1995; Lewis et al., 1996). Six of our patients had a history, of 18 months or less, of a painless mass. However, one patient had a parotid swelling for 12 years which enlarged rapidly over 7 months. Case 5 had a history of pleomorphic adenoma 20 years before, it was treated by surgery and radiotherapy. However, there was no histological evidence of a pre-existing benign mixed tumour. The histological appearance in our cases resembled ductal carcinoma of the breast, particularly the comedo-like pattern of necrosis which we found in all our cases. An intraductal component was identified in 3 tumours, although these lesions represented less than 5% of the tumour mass. Local recurrence in salivary duct carcinoma has been reported in about 45% of the cases (Brandwein et al., 1990). The incidence of local recurrence in our
series is lower. However, one patient has presented six local recurrences during an interval of 18~ years after diagnosis. Luna et al (1987) and Brandwein et al (1990) reported distant metastases in 66% of 30 patients and in 57% of 58 patients, respectively; however, in our study this incidence is lower. The presence of cervical lymph node metastases in this study is in accordance with previous reports, which ranged from 40 to 72% (Garland et al., 1984; Brandwein et al., 1990). Perineural invasion has been frequently observed (Delgado et al., 1993; Lewis et al., 1996). In our study, perineural invasion was found in 6 patients. Of our 3 patients without perineural invasion, one is alive with disease and the others are without evidence of disease 222, 24 and 3 months after surgery, respectively. Although salivary duct carcinoma was recognized as an aggressive neoplasm, in a review of previous studies (Hui et al., 1986; Afzelius et al., 1987; Brandwein et al., 1990; Simpson et al., 1991; Delgado et al., 1993; McKinney et al., 1994) a significant minority (approximately 25-30%) of patients are well with prolonged disease-free survival. However, in our short series, 6 patients are alive with or without disease. We have not found any clinicopathological feature of salivary duct carcinoma that could discriminate between those patients alive with or without disease and those dying of disease, except the presence of distant metastases. Our study shows that salivary duct carcinoma is not uncommon among malignant tumours of salivary glands, constituting up to 15% at this site. Grenko et al (1995) found 12 salivary duct carcinomas among 145 primary parotid tumours, and, in a review of 4068 salivary glands tumours, Seifert and Caselitz (1989) reported 37 cases of salivary duct carcinoma (0.9%). Taking these results into account, it is possible to suppose that this tumour could be under-recognized by those pathologists unfamiliar with it, and that regional differences in the tumour prevalence could exist. Our immunohistochemical findings, with cytokeratins (low and high molecular weight), epithelial membrane antigen and carcinoembryonic antigen positivity in tumour cells are in accordance with previous reports (Brandwein et al., 1990; Simpson et al., 1991; Delgado et al., 1993; Lewis et al., 1996). As previously described (Delgado et al., 1993; Lewis et al., 1996), our cases showed a consistent absence of S-100 protein and actin muscle-specific positivity showing that there was no evidence of myoepithelial differentiation. In addition, oestrogen receptor immunostaining was negative according to others studies (McKinney et al., 1994; Hellquist et al., 1994). Ockner et al (1994) suggested that the presence or absence of oestrogen receptor could be a distinguishing feature between salivary duct carcinoma and metastatic breast carcinoma, a hypothesis which is in agreement with our results.
Salivary duct carcinoma
In humans, several studies indicate that overexpression of c-erbB-2 oncoprotein is associated with a poor prognosis in some malignant tumours (Ro et al., 1989; Soomro et al., 1991). In salivary gland tumours, several studies have evaluated c-erbB-2 expression (Kernohan et al., 1991; Sugano et al., 1992; Barnes et al., 1994; Hellquist et al., 1994; Felix et al., 1996). Sugano et al (1992) suggested that salivary gland carcinomas showing c-erbB-2 protein positivity were aggressive in nature and had a poor prognosis, therefore these patients might benefit from a more aggressive therapy. Felix et al (1996) found that c-erbB-2 overexpression was a significant survival predictor. In others reports, c-erbB-2 overexpression ranged from 25 to 88% (Hellquist et al., 1994; Barnes et al., 1994), whereas in our study c-erbB-2 positivity did not show any significant differences between patients alive or dead from the disease. In salivary gland tumours, several studies (Soini et al., 1992; Deguchi et al., 1993) have assessed p53 immunostaining and aggressive clinical outcome. Soini et al (1992) also found correlation between DNA aneuploidy and p53 immunostaining in salivary gland tumours. In contrast, other reports (Hellquist et al., 1994; Felix et al., 1996), together with our results, have failed to find any association between p53 positivity and patient outcome. The Ki67 immunostaining reacts with a nuclear antigen present in proliferating human cells. A study using Ki67 immunostaining found high proliferative activity in this type of tumour, associated with poor patient survival (Hellquist et al., 1994). In our study the Ki67 positivity was significantly higher (> 20% of tumour cells) in patients dying of the disease. These results seem to indicate that Ki67 immunostaining can be useful in the evaluation of the biological aggressiveness of this type of tumour. DNA content in salivary gland tumours has been assessed in several studies, with differing results. Two reports describing 31 and 24 salivary duct carcinomas (Lewis et al., 1996; Felix et al., 1996) did not find any correlation between DNA ploidy or S-phase values and clinical outcome. In contrast, others studies of malignant salivary gland tumours have found correlation between DNA aneuploidy or phase fraction with the histological grade and prognosis (Hamper et al., 1989; Barnes et al., 1994). In our study, we have found a positive correlation between tumours with high proliferative index of aneuploid cells and the presence of distant metastases, as well as with fatal clinical outcome of patients. Interestingly, case 6, who is alive 222 months after diagnosis, showed an aneuploid tumour with a low proliferative index of aneuploid cells and a moderate Ki67 positivity, facts which could explain this patient's prolonged survival. In conclusion, salivary duct carcinoma may not be as rare as previously considered. Although this
333
tumour was recognized as an aggressive neoplasm, in our study six patients are alive with or without disease after 3 months, 2 years, 5 years, 11 years, 13 years and 18~ years, respectively. We think that the most effective treatment is the use of aggressive therapy (radical surgery, radical neck dissection and postoperative radiotherapy), although the results are unequal. In addition, our results seem to indicate that Ki67 immunostaining can be useful in the evaluation of the biological behaviour of these tumours, as well as the presence of a high proliferative index of aneuploid cells and the presence of distant metastases. Nonetheless, further research, with more extensive series, is needed to confirm our findings. Acknowledgements The authors thank Prof. Dr Manuel Canteras Jordana for his assistance with statistical analysis and Ms M" Dolores Lapuente, Ms Dolores Ortufio, Ms M ~ Luisa Fern/mdez, Mr Francisco C/movas and Mr Mateo Fern/mdez for their excellent technical assistance.
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Dr Enrique Martinez-Barba Pathology Service Virgen De La Arrixaca University Hospital 30120 E1Palmar Murcia Spain
Paper received 16 June 1997 Accepted 15 Oct. 1997