Eur Arch Otorhinolaryngol DOI 10.1007/s00405-015-3609-6

HEAD AND NECK

The prognostic significance of b-catenin, cyclin D1 and PIN1 in minor salivary gland carcinoma: b-catenin predicts overall survival Sven Schneider1 • Dietmar Thurnher1 • Rudolf Seemann2 • Markus Brunner1 Lorenz Kadletz1 • Bahil Ghanim3 • Klaus Aumayr4 • Gregor Heiduschka1 • Claudia Lill1



Received: 21 December 2014 / Accepted: 17 March 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Minor salivary gland carcinoma is a rare and heterogeneous type of cancer. Molecular prognostic and predictive markers are sparse. The aim of this study was to identify new prognostic and predictive markers in minor salivary gland carcinoma. 50 tissue samples of carcinomas of the minor salivary glands (adenoid cystic carcinoma n = 23, mucoepidermoid carcinoma n = 12, adenocarcinoma n = 10, carcinoma ex pleomorphic adenoma n = 2, salivary duct carcinoma n = 1, clear cell carcinoma n = 1, basal cell carcinoma n = 1) were immunohistochemically stained for b-catenin, cyclin D1 and PIN1. Expression patterns were analyzed and correlated to clinical outcome of 37 patients with complete clinical data. High expression of membranous b-catenin was linked to significantly better overall survival in patients with adenoid cystic carcinoma (log rank test, v2 = 13.3, p = .00397, Bonferroni corrected p = .024). PIN1 and cyclin D1 did not show any significant correlation to patients’ clinical outcome. Expression of b-catenin in adenoid cystic carcinoma of the minor salivary glands significantly correlates with better overall survival. Hence, evaluation of b-catenin might serve as a clinical prognostic marker.

& Gregor Heiduschka [email protected] 1

Department of Otorhinolaryngology-Head and Neck Surgery, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria

2

Department of Cranio-, Maxillofacial and Oral Surgery, Medical University of Vienna, Vienna, Austria

3

Division of Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria

4

Department of Pathology, Medical University of Vienna, Vienna, Austria

Keywords b-Catenin  Cyclin D1  PIN1  Minor salivary gland cancer  Molecular prognostic markers

Introduction Minor salivary gland carcinoma is a rare neoplasia of the upper aero-digestive tract with a wide range of possible pathohistological diagnoses [1]. The majority of minor salivary glands are located in the oral cavity and pharynx, although they can also be located in the middle ear, the paranasal sinuses and the larynx. Surgery and adjuvant radiotherapy is the most frequently used therapeutic approach, whereby 5-year survival rates vary from 66 to 80 % [2–4]. Clinicopathological prognostic factors include TNM classification, grading and surgical margins as well as anatomic site as independent markers [5–8]. Also, molecular markers such as growth factor receptor proteins, cell cycle oncogenes and proteins involved in apoptosis and cell–cell adhesion have been evaluated for prognostic potential, and some of them have additional prognostic value [9–12]. Despite gaining further insights into the molecular background in this rare malignancy, the mechanisms of malignant progression and differential behavior in the particular tumor entities are not yet clear. The b-catenin protein is involved in the regulation of cell–cell adhesion and plays a major role in Wnt signaling transduction acting as a transcription cofactor with T cell/lymphoid enhancer factor [13]. After initiation of Wnt signaling by binding of Wnt protein to a receptor of the Frizzled family, b-catenin accumulates in the cytoplasm and eventually migrates to the nucleus [14]. Aberrant expression of b-catenin is described in various types of cancer including colorectal cancer, lung cancer, malignant breast tumors and head and neck squamous cell carcinoma

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[15–18]. Furthermore, there is evidence of abnormal expression in salivary gland carcinoma [19, 20]. Accumulation of b-catenin bound to transcription cofactor T cell factor/lymphoid enhancing factor can regulate target gene expression in the nucleus. A direct target of this complex is cyclin D1, a critical cell cycle regulator containing a lymphoid enhancing factor binding sequence [21]. Cyclin D1 induces G1 to S phase transition and therefore promotes proliferation and possible invasiveness of cancer cells. Aberrant cyclin D1 expression has been observed in various types of cancer, including breast, esophageal and thyroid cancer as well as head and neck squamous cell carcinoma [22–26]. Activation of b-catenin is regulated by epigenetic factors, such as Pin1, a peptidyl-prolyl cis–trans isomerase enzyme. Pin1 specifically regulates phosphorylated serine/threonine residues preceding proline peptide bonds and directly binds a phosphorylated Ser-Pro motif next to the APC-binding site in b-catenin [27, 28]. Subsequently, b-catenin dissociates from APC-binding complex, accumulates in the cytoplasm and translocates to the nucleus where cyclin D1 expression is activated. Furthermore, Pin1 can bind Ser-Pro motifs in c-Jun, increasing the ability of c-Jun to activate the cyclin D1 promoter [27]. Aberrant Pin1 expression itself was observed in various types of cancer such as esophageal, breast and Merkel cell carcinoma [29–31]. Recent studies have shown that protein expression analysis via immunohistochemistry provides interesting insights into molecular mechanisms in various types of cancer including salivary gland carcinoma and head and neck squamous cell carcinoma. Furthermore, correlation of immunohistochemical staining patterns to patients’ clinical data reveals promising molecular markers regarding patients’ prognosis [11, 31–34]. This study investigates the expression of b-catenin, cyclin D1 and Pin1 in carcinomas of the minor salivary glands and their prognostic relevance for patients’ survival.

Materials and methods Patients In this retrospective study, tissue samples of 50 patients with minor salivary gland carcinoma were included. Patients were diagnosed and treated between 1970 and 2013 at the Medical University of Vienna. This study included only patients with newly diagnosed minor salivary gland cancer who were subsequently followed at our institution. The detailed histological classification of tumor samples is shown in Table 1. Patients with incomplete medical records (11 patients), primary R ? resection (2 patients) and patients with

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Table 1 Cases used for immunohistochemical analysis 50

Minor salivary gland carcinomas

23

Adenoid cystic carcinomas

12

Mucoepidermoid carcinomas

10

Adenocarcinomas

2

Carcinomas ex pleomorphic adenoma

1

Salivary duct carcinoma

1

Clear cell carcinoma

1

Basal cell carcinoma

missing tissue samples due to tissue float off during the immunohistochemical staining process were excluded from statistical analysis. Detailed information for the patients included in statistical analysis is shown in Table 2. Approval for the study was obtained from the institutional Research Ethics Board (EK-NR 912/2010). Clinical data In summary, tumor samples of 50 patients with minor salivary gland carcinoma were investigated for expression of b-catenin, cyclin D1 and PIN1 via immunohistochemical staining. For statistical analysis, 13 patients had to be excluded due to insufficient data. In the remaining group of 37 patients, 21 patients were female and 16 male with a mean age of 55 years (median 59 years, range 25–87 years). At diagnosis, 18 patients presented in UICC (Union internationale contre le cancer) I stage, 7 patients in UICC II stage, 6 patients in UICC III stage and 6 in UICC IV stage. At the end of the study (June 1st, 2014), 18 patients were alive without disease, 3 patients were alive with disease and 16 patients have died of disease. The mean range of follow-up was 101 months (median 105 months, range 1–296 months) and mean disease-free survival was 78 months (median 47 months, range 1–252 months). The 5-year overall survival rate for all malignant tumors was 74 %. Stratified for histopathological subtype, the 5-year overall survival rate for adenoid cystic carcinoma was 62 %, mucoepidermoid carcinoma 91 %, and adenocarcinoma patients 60 %. Clinical data are listed in Table 2. Immunohistochemistry Immunohistochemistry was performed using Ultravision LP detection system HRP Polymer (RTU) (Thermo Scientific, Fremont, CA, USA) according to the manufacturer’s protocol. Briefly, dewaxed and rehydrated tumor sections (2–3 lm thickness) were subjected to antigen retrieval in a

Eur Arch Otorhinolaryngol Table 2 Clinical data for patients included in statistical analysis Patient no.

Localization

Histology

PIN1

Cy D

b cat

UICC

T classification

N classification

M classification

Treatment

1

Mandible

Adeno CA

2

1

3

I

T1

N0

M0

OP ? ND

2

Hard palate

MEC

lot

1

lot

I

T1

N0

M0

OP

4

Mandible

MEC

lot

2

3

II

T2

N0

M0

OP ? ND

5

Hard palate

ACC

lot

1

lot

I

T1

N0

M0

OP

7

Hard palate

BC adeno CA

1

2

3

I

T1

N0

M0

OP

8

Tongue

MEC

lot

lot

lot

II

T2

N0

M0

pRCT

10

Hard palate

Adeno CA

1

1

3

I

T1

N0

M0

OP

11

Base of tongue

MEC

1

0

2

III

T2

N1

M0

OP ? ND ? PORT

12

Hard palate

CA ex PL

1

1

1

II

T2

N0

M0

OP ? ND

13

Mandible

MEC

1

2

1

I

T1

N0

M0

OP

14

Hard palate

Adeno CA

2

2

1

I

T1

N0

M0

OP

15

Nasal cavity

ACC

lot

0

2

III

T3

N0

M0

OP ? PORT

18

External auditory canal

Adeno CA

2

2

1

I

T1

N0

M0

OP

19 21

Soft palate Hard palate

ACC ACC

2 lot

2 lot

3 0

II IV

T2 T3

N0 N0

M0 M1 (pul)

OP ? PORT pall RT

23

Base of tongue

MEC

1

1

1

III

T2

N1

M0

OP ? ND

24

Hard palate

ACC

0

1

3

I

T1

N0

M0

OP

25

Larynx

Adeno CA

2

1

1

II

T2

N0

M0

OP

26

Hard palate

ACC

lot

lot

lot

IV

T4a

N0

M0

OP ? PORT

27

Base of tongue

ACC

3

1

3

II

T2

N0

M0

OP

30

Hard palate

CA ex PL

1

1

1

I

T1

N0

M0

OP

32

Sinus

ACC

3

1

1

III

T3

N0

M0

OP ? PORT

34

Upper lip

MEC

1

2

2

I

T1

N0

M0

OP

35

Hard palate

MEC

lot

1

2

I

T1

N0

M0

OP

36

Hard palate

ACC

2

2

3

IV

T4a

2b

M0

OP ? PORT

38

Cheek

MEC

1

2

2

I

T1

N0

M0

OP

39

Sinus

ACC

2

1

2

III

T3

N0

M0

OP ? PORT

40

Hard palate

MEC

2

2

2

IV

T4a

N0

M0

OP

41 42

Soft palate Larynx

Adeno CA MEC

1 1

2 1

1 2

I I

T1 T1

N0 N0

M0 M0

OP OP

44

Hard palate

MEC

2

2

2

I

T1

N0

M0

OP

45

Cheek

ACC

lot

1

lot

I

T1

N0

M0

OP

46

Floor of the mouth

Adeno CA

1

1

2

I

T1

N0

M0

OP

47

Hard palate

Adeno CA

2

2

1

IV

T3

N2b

M0

OP ? ND

48

Cheek

ACC

2

2

1

III

T3

N0

M0

OP

49

Sinus

ACC

0

1

2

IV

T4b

N0

M0

pall OP ? RT

50

Tongue

ACC

2

2

2

II

T2

N0

M0

pRT

Adeno CA adenocarcinoma, ACC adenoid cystic carcinoma, BC adeno CA basal cell adenocarcinoma, CA ex PL carcinoma ex pleomorphic adenoma, MEC mucoepidermoid carcinoma, lot loss of tissue, OP surgery, PORT post operative radiotherapy, pRT primary radiotherapy, Pall OP palliative surgery, Pall RT palliative radiotherapy

microwave oven (600 W) employing citrate buffer. Endogenous peroxidase activity was blocked with 3 % H2O2 for 15 min. After incubation with Ultra V Block for 5 min, the primary antibody was applied at room temperature for 1 h. Antibodies directed against b-catenin (dilution 1:100,

Santa Cruz Biotechnology, Dallas, Texas, USA) cyclin D1 (dilution 1:200, Invitrogen, Camarillo, CA, USA) and PIN1 (dilution 1:100, Abcam, Cambridge, UK) were used in this study. Next, Primary Antibody Enhancer was applied for 10 min followed by HRP Polymer for 15 min.

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Visualization was performed by UltraVision Plus Detection System DAB Plus Substrate System (RTU) (Thermo Scientific, Fremont, CA, USA). Consecutively, slides were counterstained with Hematoxylin Gill III (Merck, Darmstadt, Germany). Samples were analyzed using an Olympus BH-2 microscope (Olympus America, Center Valley, PA). Negative controls were performed by replacement of the primary antibody with rabbit immunoglobulin G isotype control (Abcam, rabbit polyclonal immunoglobulin G, 1:200, citrate buffer), following the same protocol. Positive controls were performed according to the manufacturer’s protocol. Specimen classification based on immunohistochemical results and morphological features Protein expression of b-catenin, cyclin D1 and PIN1 was determined in the cytoplasm, membrane and the nucleus of the tumor. All slides were assigned to one of the four categories of marker expression: 0: absence of reactivity in all neoplastic cells; 1 (weak): focal staining in some of the neoplastic cells (\10 %); 2 (moderate): more extensive discontinuous staining still not fitting the criteria for continuously staining in the majority of the neoplastic cells (10–80 %); 3 (strong): entire and continuous staining in all neoplastic cells. If there were two patterns in a case, the higher staining intensity number was considered to be representative. All cases were reviewed by an experienced pathologist (A. K.). Observer bias was avoided by repeating the evaluation of protein expression at two different time points and without knowledge of patients’ clinical data. Statistical analysis The overall survival was defined as the period from patients’ first treatment until death or end of the study (June 1, 2014). The disease-free interval was defined as the period from patients’ first treatment until recurrence of disease or from patients’ first treatment to the end of the study. The overall survival and disease-free interval curves were estimated using the Kaplan–Meier method and analyzed regarding b-catenin, cyclin D1 and PIN1 using log rank tests. Bonferroni correction was calculated to avoid the error rate for multiple testing. A value of p \ .05 was considered statistically significant. Statistical analysis was carried out with the open source statistical programming environment ‘‘R’’, version 2.15.1 (The R Foundation for Statistical Computing, Vienna, Austria).

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Results Immunohistochemistry Representative tissue samples of 50 patients with minor salivary gland carcinoma were immunohistochemically stained with antibodies directed against b-catenin, cyclin D1 and PIN1. All proteins were expressed in the cytoplasm or the membrane of the cancer cells. b-Catenin staining was predominantly membranous, whereas staining PIN1 and cyclin D1 showed cytoplasmatic staining. Expression patterns are shown in Fig. 1 and Table 3. Evaluation of expression patterns in all tissue samples b-Catenin expression was observed in 41 (93 %) of 44 samples. Weak expression was detected in 14 (32 %), moderate expression in 17 (39 %) and strong expression in 10 (23 %) of investigated samples. 3 samples (7 %) showed no staining. Cyclin D1 expression could be shown in 45 (96 %) of 47 samples. Expression pattern was classified weak in 26 (55 %), moderate in 19 (40 %) and strong in 0 (0 %) of analyzed samples, whereas no expression was found in 2 (4 %) cases. PIN1 expression was detected in 37 (95 %) of 39 samples. Weak expression was found in 17 (44 %), moderate expression in 17 (44 %) and strong expression in 3 (8 %) of analyzed samples, whereas no expression was detected in 2 (5 %) cases. Evaluation of expression patterns stratified for histopathological classification Adenoid cystic carcinoma In adenoid cystic carcinoma, b-catenin expression was observed in 17 (89 %) of 19 samples. Expression was classified weak in 4 (21 %), moderate in 7 (37 %) and strong in 6 (32 %) cases. 2 (11 %) showed no staining. Cyclin D1 expression was observed in 20 (95 %) of 21 samples. Weak expression was detected in 13 (62 %), moderate in 7 (33 %) and strong expression in 0 (0 %) of cases, whereas 1 (5 %) showed no staining. Staining for PIN1 could be shown in 14 (88 %) of 16 tissue samples. 3 (19 %) cases showed weak, 8 (50 %) moderate and 3 (19 %) strong expression, whereas 2 (13 %) showed no staining. Adenocarcinoma b-Catenin expression was detected in 9 (90 %) of 10 samples. 6 (60 %) cases showed weak, 1 (10 %) moderate and 2 (20 %) strong expression, whereas 1 (10 %) showed no staining.

Eur Arch Otorhinolaryngol Fig. 1 Immunohistochemical staining of minor salivary gland carcinomas. Positive cyclin D1 staining (a), negative cyclin D1 staining (b), positive PIN1 staining (c), negative PIN1 staining (d), positive b-catenin staining (e), negative b-catenin staining (f). All pictures taken at 9200 magnification

Cyclin D1 protein staining was observed in 10 (100 %) of 10 samples. 6 (60 %) samples showed weak and 4 (40 %) moderate expression. No sample showed negative or strong expression. PIN1 expression was found in 10 (100 %) of 10 samples, whereas weak expression was detected in 4 (40 %) and moderate in 6 (60 %). No sample showed negative or strong expression.

PIN1 staining could be observed in 8 (100 %) of 8 cases. 6 (75 %) samples showed weak and 2 (25 %) moderate expression. Strong or negative staining was not detected.

Mucoepidermoid carcinoma b-Catenin expression 10 (100 %) of 10 samples, whereas staining was classified weak in 2 (20 %), moderate in 7 (70 %) and strong in 1 (10 %) cases. Negative expression was not observed. Cyclin D1 expression was found in 10 (91 %) of samples, whereas 4 (36 %) showed weak, 6 (55 %) showed moderate and 0 (0 %) strong staining. No expression was detected in 1 (9 %) case.

Basal cell adenocarcinoma 1 (100 %) sample of basal cell adenocarcinoma showed strong expression for bcatenin, moderate expression for cyclin D1 and weak expression for PIN1.

Carcinoma ex pleomorphic adenoma In 2 (100 %) samples of carcinoma ex pleomorphic adenoma expression was classified weak for b-catenin, cyclin D1 and PIN1.

Clear cell carcinoma In 1 (100 %) sample of clear cell carcinoma expression was classified moderate for bcatenin, cyclin D1 and PIN1.

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Eur Arch Otorhinolaryngol Table 3 Schematic representation of immunohistochemical staining intensity in minor salivary gland carcinomas Patient no.

Histo

PIN1

Cy D

b cat

1

Adeno CA

2

1

3

2

MEC

lot

1

lot

3

Adeno CA

2

1

1

4

MEC

lot

2

3

5

ACC

lot

1

lot

6

ACC

1

1

1

7 8

BC Adeno CA MEC

1 lot

2 lot

3 lot

9

ACC

lot

1

lot

10

Adeno CA

1

1

3

11

MEC

1

0

2

12

CA ex PL

1

1

1

13

MEC

1

2

1

14

Adeno CA

2

2

1

15

ACC

lot

0

2

16

Adeno CA

1

1

0

17

ACC

3

2

3

18

Adeno CA

2

2

1

19

ACC

2

2

3

20

ACC

1

1

2

21

ACC

lot

lot

0

22 23

CCC MEC

2 1

2 1

2 1

24

ACC

0

1

3

25

Adeno CA

2

1

1

26

ACC

lot

lot

lot

27

ACC

3

1

3

28

ACC

lot

2

1

29

ACC

2

1

2

30

CA ex PL

1

1

1

31

ACC

2

2

3

32

ACC

3

1

1

33

ACC

1

1

0

34

MEC

1

2

2

35

MEC

lot

1

2

36

ACC

2

2

3

37

ACC

2

1

2

38 39

MEC ACC

1 2

2 1

2 2

40

MEC

2

2

2

41

Adeno CA

1

2

1

42

MEC

1

1

2

43

SDC

1

1

2

44

MEC

2

2

2

45

ACC

lot

1

lot

46

Adeno CA

1

1

2

47

Adeno CA

2

2

1

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Table 3 continued Patient no.

Histo

PIN1

Cy D

b cat

48

ACC

2

2

1

49

ACC

0

1

2

50

ACC

2

2

2

Cy D cyclin D1, b cat b-catenin, Adeno CA adenocarcinoma, ACC adenoid cystic carcinoma, BC Adeno CA basal cell adenocarcinoma, CA ex PL carcinoma ex pleomorphic adenoma, MEC mucoepidermoid carcinoma, SDC salivary duct carcinoma

Fig. 2 Kaplan–Meier curve of the influence of the Union Internationale Contre le Cancer (UICC) stage on overall survival of patients

Salivary duct carcinoma In 1 (100 %) sample of salivary duct carcinoma expression was classified moderate for bcatenin staining. PIN1, cyclin D1 showed weak staining. Correlation with patients’ survival data Statistical correlation of protein expression with patients’ clinical data was performed for all cases as well as stratified for histopathological diagnoses. In all cases, not regarding histopathological diagnosis, analysis of overall survival, disease-free survival and UICC stage did not reveal any statistically significant correlation with b-catenin, cyclin D1 or PIN1 protein expression, although a clear trend for better overall survival in patients with lower UICC staging could be observed. Kaplan–Meier curves for UICC stages are shown in Fig. 2. Subgroup analysis was performed for adenoid cystic carcinoma. For adenocarcinoma, mucoepidermoid carcinoma, carcinoma ex pleomorphic adenoma, basal cell adenocarcinoma, clear cell carcinoma and salivary duct carcinoma, no statistical calculation was performed due to small sample size or missing clinical data.

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In adenoid cystic carcinoma, the log rank test showed statistically significant correlation (log rank test, v2 = 13.3, p = .00397, Bonferroni corrected p = .024) for b-catenin expression linked to overall survival. No significant correlation was observed for disease-free survival and b-catenin expression (log rank test, v2 = 4.5, p = .215, Bonferroni corrected p = 1.0). Kaplan–Meier curves for b-catenin expression and patients’ disease-free and overall survival are shown in Figs. 3 and 4. Expression of PIN1 (overall survival: log rank test, v2 = 0.2, p = .899, Bonferroni corrected p = 1.0; diseasefree survival: log rank test, v2 = 0.1, p = .943, Bonferroni

Fig. 3 Kaplan–Meier curve of the influence of b-catenin on diseasefree survival (DFS) of patients

Fig. 4 Kaplan–Meier curve of the influence of b-catenin on overall survival of patients. The expression of b-catenin is an independent prognostic marker for overall survival of patients with adenoid cystic minor salivary gland carcinoma (p = 0.00397; Bonferroni corrected p = 0.024)

corrected p = 1.0) and cyclin D1 (overall survival: log rank test, v2 = 0.3, p = .848, Bonferroni corrected p = 1.0; disease-free survival: log rank test, v2 = 1.7, p = .431, Bonferroni corrected p = 1.0) did not show any statistically significant correlation with overall survival or disease-free survival.

Discussion Minor salivary gland carcinoma is a rare type of cancer. In this study, we evaluated an extensive and representative group of patients. Stratification for histopathological subtype and correlation to patients’ clinical outcome revealed significant correlation of b-catenin expression to overall survival in adenoid cystic carcinoma. Patients’ prognosis is not only dependent on histopathological diagnosis, but also primary tumor site regarding, minor or major salivary glands as the place of origin [35]. In recent studies, independent prognostic factors for disease-free and disease-specific survival, including tumor size, grade, surgical margins, perineural invasion and lymph node status, have been described [5, 36]. In contrast to head and neck squamous cell carcinoma, human papilloma virus infection and p16 protein expression do not have any prognostic or predictive relevance [37]. Studies investigating a combined set of minor and major salivary gland carcinoma could specify molecular prognostic markers including Ki-67, EGFR and C-KIT [11, 38]. However, there is a lack of molecular markers specific for minor salivary gland carcinoma, stratified for histopathological diagnoses. In this study, cyclin D1 expression was frequently detected in most of the investigated tissue samples, whereupon similar expression patterns have been shown previously in adenoid cystic carcinoma [39]. Confirming previous investigations, expression patterns of cyclin D1 and PIN1 did neither reveal any prognostic significance in adenoid cystic carcinoma nor in the complete collective. Similar findings were described in a mixed cohort of adenoid cystic carcinomas of the minor and major salivary glands, whereas no correlation with clinicopathological parameters could be observed [19]. Furthermore, no association between cyclin D1 expression and cell proliferation could be determined in vitro and, moreover, cyclin D1 expression is not correlated to an unfavorable outcome in patients with adenoid cystic salivary gland carcinoma in a collective of parotid gland, submandibular gland and minor salivary gland carcinomas [40]. Expression of PIN1 is frequently reported in various types of cancer and linked to tumor proliferation and prognosis [29–31]. Interestingly, studies evaluating PIN1 expression in salivary gland carcinoma are rare. We could

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show protein expression in the majority of tissue samples. However, no correlation to patients’ clinical outcome could be determined. Similar expression patterns have been described before in a mixed cohort of minor and major salivary gland carcinomas, whereas no correlation to clinicopathological parameters could be revealed and correlation to survival data was not performed [19]. In a recent study, PIN1 expression could be observed in almost all tumor samples of an extensive cohort of parotid gland carcinomas, although no correlation to patients’ outcome could be observed [31]. In this study, we found PIN1 expression in carcinoma of the minor salivary glands, yet, the relevance for carcinogenesis and proliferation has to be determined in further investigations. b-Catenin has multiple functions in various kinds of malignant tumors. Recent studies have shown that expression of b-catenin is linked to patients’ clinical outcome, whereby the differentiation of membranous and nuclear staining has to be made. In mucoepidermoid carcinoma of the minor and major salivary glands, it has been reported that nuclear b-catenin expression is mainly found in patients with an unfavorable prognosis [41]. Furthermore, cytoplasmatic b-catenin expression is a possible prognostic marker in parotid gland cancer [31]. Cytoplasmatic b-catenin expression correlated significantly with an adverse disease-free and overall survival in all parotid gland carcinomas, not regarding pathohistological diagnosis [31]. Similar results were observed in a subset of mucoepidermoid carcinoma of the salivary glands. Cytoplasmatic accumulation was seen in patients with poor overall survival, making the authors emphasize that bcatenin location could be a useful predictor of tumor progression and prognosis [42]. However, a study assessing the expression in benign compared to malignant salivary gland tumors described reduced membranous and predominant cytoplasmic localization of b-catenin expression in salivary gland carcinomas, indicating a lack of differentiation and higher invasive potential [43]. In line with this report are similar findings in adenoid cystic carcinomas, whereas reduced membranous expression was found in most patients with metastasis and correlated significantly with nodal metastasis [19]. In mucoepidermoid carcinoma, aberrant b-catenin expression, by the means of increased or decreased expression, is linked to an unfavorable overall survival [20]. Not only in salivary gland carcinoma, but also in non-small-cell lung cancer high levels of membranous b-catenin expression indicate a better prognosis [16]. In this study, expression of b-catenin was observed in 93 % of all tumor samples and 89 % of adenoid cystic carcinomas, obtaining a widespread field of staining intensities in the range of no to strong expression. Similar expression patterns have been described before, whereas

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positive staining was found in 67.4 % in a collective of mixed histopathological diagnoses of salivary gland cancer and in up to 100 % of adenoid cystic carcinomas [31, 44, 45]. Although there is evidence for aberrant b-catenin expression in salivary gland cancer, so far no study investigated the impact of b-catenin on patients’ survival in adenoid cystic carcinoma of the minor salivary glands. A possible limitation of this study is that protein expression was assessed in tissue samples only on protein level by immunohistochemistry as a single method. Although the authors believe that semiquantitative analysis by pathologist visual scoring is an appropriate approach to determine protein expression, further studies investigating transcriptional levels of the investigated proteins by polymerase chain reaction analysis and subsequent measurement of protein quantity by enzyme-linked immunosorbent assays are of great interest [46]. In this study, we could show that high expression of membranous b-catenin is significantly correlated to a better overall survival in patients with minor salivary gland adenoid cystic carcinoma. Regarding these results, evaluation of b-catenin staining might serve as a promising molecular marker in this rare type of cancer. Conflict of interest of interest.

The authors declare that they have no conflict

References 1. Barnes L, Eveson JW, Reichart P, Sidransky D (2005) World Health Organization classification of tumors. Pathology and genetics of head and neck tumors. IARC Press, Lyon, pp 209–281 2. Vander Poorten VL, Balm AJ, Hilgers FJ et al (2000) Stage as major long term outcome predictor in minor salivary gland carcinoma. Cancer 89:1195–1204 3. Lopes MA, Santos GC, Kowalski LP (1998) Multivariate survival analysis of 128 cases of oral cavity minor salivary gland carcinomas. Head Neck 20:699–706 4. Jones AS, Beasley NJ, Houghton DJ et al (1998) Tumours of the minor salivary glands. Clin Otolaryngol Allied Sci 23:27–33 5. Carrillo JF, Maldonado F, Carrillo LC et al (2011) Prognostic factors in patients with minor salivary gland carcinoma of the oral cavity and oropharynx. Head Neck 33:1406–1412. doi:10.1002/ hed.21641 6. Beckhardt RN, Weber RS, Zane R et al (1995) Minor salivary gland tumors of the palate: clinical and pathologic correlates of outcome. Laryngoscope 105:1155–1160. doi:10.1288/00005537199511000-00003 7. Kakarala K, Bhattacharyya N (2010) Survival in oral cavity minor salivary gland carcinoma. Otolaryngol Head Neck Surg 143:122–126. doi:10.1016/j.otohns.2010.02.033 8. Cianchetti M, Sandow PS, Scarborough LD et al (2009) Radiation therapy for minor salivary gland carcinoma. Laryngoscope 119:1334–1338. doi:10.1002/lary.20501 9. Cho KJ, Lee SS, Lee YS (1999) Proliferating cell nuclear antigen and c-erbB-2 oncoprotein expression in adenoid cystic carcinomas of the salivary glands. Head Neck 21:414–419

Eur Arch Otorhinolaryngol 10. Luukkaa H, Klemi P, Leivo I et al (2006) Prognostic significance of Ki-67 and p53 as tumor markers in salivary gland malignancies in Finland: an evaluation of 212 cases. Acta Oncol 45:669–675. doi:10.1080/02841860500543208 11. Ettl T, Schwarz S, Kleinsasser N et al (2008) Overexpression of EGFR and absence of C-KIT expression correlate with poor prognosis in salivary gland carcinomas. Histopathology 53:567–577. doi:10.1111/j.1365-2559.2008.03159.x 12. Stenner M, Klussmann JP (2009) Current update on established and novel biomarkers in salivary gland carcinoma pathology and the molecular pathways involved. Eur Arch Otorhinolaryngol 266:333–341. doi:10.1007/s00405-008-0882-7 13. Behrens J, von Kries JP, Ku¨hl M et al (1996) Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 382:638–642. doi:10.1038/382638a0 14. Rao TP, Ku¨hl M (2010) An updated overview on Wnt signaling pathways: a prelude for more. Circ Res 106:1798–1806. doi:10. 1161/CIRCRESAHA.110.219840 15. Morin PJ, Sparks AB, Korinek V et al (1997) Activation of betacatenin-Tcf signaling in colon cancer by mutations in betacatenin or APC. Science 275:1787–1790 16. Retera JM, Leers MP, Sulzer MA, Theunissen PH (1998) The expression of beta-catenin in non-small-cell lung cancer: a clinicopathological study. J Clin Pathol 51:891–894 17. Lin SY, Xia W, Wang JC et al (2000) Beta-catenin, a novel prognostic marker for breast cancer: its roles in cyclin D1 expression and cancer progression. Proc Natl Acad Sci USA 97:4262–4266. doi:10.1073/pnas.060025397 18. Yang F, Zeng Q, Yu G et al (2006) Wnt/beta-catenin signaling inhibits death receptor-mediated apoptosis and promotes invasive growth of HNSCC. Cell Signal 18:679–687. doi:10.1016/j.cell sig.2005.06.015 19. Zhou C-X, Gao Y (2006) Aberrant expression of beta-catenin, Pin1 and cyclin D1 in salivary adenoid cystic carcinoma: relation to tumor proliferation and metastasis. Oncol Rep 16:505–511 20. Shieh Y-S, Chang L-C, Chiu K-C et al (2003) Cadherin and catenin expression in mucoepidermoid carcinoma: correlation with histopathologic grade, clinical stage, and patient outcome. J Oral Pathol Med 32:297–304 21. Shtutman M, Zhurinsky J, Simcha I et al (1999) The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci USA 96:5522–5527 22. Khramtsov AI, Khramtsova GF, Tretiakova M et al (2010) Wnt/beta-catenin pathway activation is enriched in basal-like breast cancers and predicts poor outcome. Am J Pathol 176:2911–2920. doi:10.2353/ajpath.2010.091125 23. Zhao J, Li L, Wei S et al (2012) Clinicopathological and prognostic role of cyclin D1 in esophageal squamous cell carcinoma: a meta-analysis. Dis Esophagus 25:520–526. doi:10.1111/j.14422050.2011.01278.x 24. Nakashima M, Meirmanov S, Naruke Y et al (2004) Cyclin D1 overexpression in thyroid tumours from a radio-contaminated area and its correlation with Pin1 and aberrant beta-catenin expression. J Pathol 202:446–455. doi:10.1002/path.1534 25. Rasamny JJ, Allak A, Krook KA et al (2012) Cyclin D1 and FADD as biomarkers in head and neck squamous cell carcinoma. Otolaryngol Head Neck Surg 146:923–931. doi:10.1177/ 0194599811435052 26. Higuchi E, Oridate N, Homma A et al (2007) Prognostic significance of cyclin D1 and p16 in patients with intermediate-risk head and neck squamous cell carcinoma treated with docetaxel and concurrent radiotherapy. Head Neck 29:940–947. doi:10. 1002/hed.20632 27. Ryo A, Nakamura M, Wulf G et al (2001) Pin1 regulates turnover and subcellular localization of beta-catenin by inhibiting its

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

interaction with APC. Nat Cell Biol 3:793–801. doi:10.1038/ ncb0901-793 Tsuchiya R, Yamamoto G, Nagoshi Y et al (2004) Expression of adenomatous polyposis coli (APC) in tumorigenesis of human oral squamous cell carcinoma. Oral Oncol 40:932–940. doi:10. 1016/j.oraloncology.2004.04.011 Fukuchi M, Fukai Y, Kimura H et al (2006) Prolyl isomerase Pin1 expression predicts prognosis in patients with esophageal squamous cell carcinoma and correlates with cyclin D1 expression. Int J Oncol 29:329–334 Wulf GM, Ryo A, Wulf GG et al (2001) Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1. EMBO J 20:3459–3472. doi:10.1093/emboj/20.13.3459 Lill C, Schneider S, Pammer J et al (2011) Significant correlation of peptidyl-prolyl isomerase overexpression in Merkel cell carcinoma with overall survival of patients. Head Neck 33:1294– 1300. doi:10.1002/hed.21596 Toyoda M, Kaira K, Shino M et al (2014) CD98 as a novel prognostic indicator for patients with stage III/IV hypopharyngeal squamous cell carcinoma. Head Neck. doi:10.1002/hed. 23797 Lill C, Schneider S, Seemann R et al (2013) Correlation of bcatenin, but not PIN1 and cyclin D1, overexpression with diseasefree and overall survival in patients with cancer of the parotid gland. Head Neck. doi:10.1002/hed.23546 Murakami N, Mori T, Yoshimoto S et al (2014) Expression of EpCAM and prognosis in early-stage glottic cancer treated by radiotherapy. Laryngoscope 124:E431–E436. doi:10.1002/lary. 24839 Marcinow A, Ozer E, Teknos T et al (2013) Clinicopathologic predictors of recurrence and overall survival in adenoid cystic carcinoma of the head and neck: a single institutional experience at a tertiary care center. Head Neck. doi:10.1002/hed.23523 Loh KS, Barker E, Bruch G et al (2009) Prognostic factors in malignancy of the minor salivary glands. Head Neck 31:58–63. doi:10.1002/hed.20924 Brunner M, Koperek O, Wrba F et al (2012) HPV infection and p16 expression in carcinomas of the minor salivary glands. Eur Arch Otorhinolaryngol 269:2265–2269. doi:10.1007/s00405-0111894-2 Larsen SR, Bjørndal K, Godballe C, Krogdahl A (2012) Prognostic significance of Ki-67 in salivary gland carcinomas. J Oral Pathol Med 41:598–602. doi:10.1111/j.1600-0714.2012. 01148.x Greer RO, Said S, Shroyer KR et al (2007) Overexpression of cyclin D1 and cortactin is primarily independent of gene amplification in salivary gland adenoid cystic carcinoma. Oral Oncol 43:735–741. doi:10.1016/j.oraloncology.2006.09.007 Yasumatsu R, Kuratomi Y, Nakashima T et al (2004) Cyclin D1 expression does not effect cell proliferation in adenoid cystic carcinoma of the salivary gland. Eur Arch Otorhinolaryngol 261:526–530. doi:10.1007/s00405-003-0724-6 Shiratsuchi H, Nakashima T, Hirakawa N et al (2007) betaCatenin nuclear accumulation in head and neck mucoepidermoid carcinoma: its role in cyclin D1 overexpression and tumor progression. Head Neck 29:577–584. doi:10.1002/hed.20583 Lee C-H, Hung Y-J, Lin C-Y et al (2010) Loss of SFRP1 expression is associated with aberrant beta-catenin distribution and tumor progression in mucoepidermoid carcinoma of salivary glands. Ann Surg Oncol 17:2237–2246. doi:10.1245/s10434-0100961-z Chandrashekar C, Angadi PV, Krishnapillai R (2011) b-Catenin expression in benign and malignant salivary gland tumors. Int J Surg Pathol 19:433–440. doi:10.1177/1066896909346366

123

Eur Arch Otorhinolaryngol 44. Daa T, Kaku N, Kashima K et al (2005) Expression of betacatenin, E-cadherin and cyclin D1 in adenoid cystic carcinoma of the salivary gland. J Exp Clin Cancer Res 24:83–87 45. Furuse C, Cury PR, Altemani A et al (2006) Beta-catenin and E-cadherin expression in salivary gland tumors. Int J Surg Pathol 14:212–217. doi:10.1177/1066896906290652

123

46. Rizzardi AE, Johnson AT, Vogel RI et al (2012) Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. Diagn Pathol 7:42. doi:10.1186/1746-1596-7-42

The prognostic significance of β-catenin, cyclin D1 and PIN1 in minor salivary gland carcinoma: β-catenin predicts overall survival.

Minor salivary gland carcinoma is a rare and heterogeneous type of cancer. Molecular prognostic and predictive markers are sparse. The aim of this stu...
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