Supplement

Supplemental Text

Chromosomal imbalances in salivary gland acinar cell carcinomas

Aside from PACs, carcinomas of acinar differentiation occur in the salivary glands, especially in the parotid gland (80%) and in intraoral minor salivary glands (17%) [1]. Salivary gland acinar cell carcinomas (SGACs) may affect patients at any age, showing an even sex distribution [1]. The prognosis of SGACs is more favourable than of PACs, low rates of metastases and disease-associated death (16%) have been reported[1]. Phenotypically, SGAC display serous acinar differentiation which can be reproduced by immunohistochemical positivity for amylase in most cases [1].

In this study, acinar cell carcinoma samples of salivary glands were obtained from 6 women and 6 men at ages between 27 and 77 years (mean 56.2 years). The 13 samples consisted of 9 primary tumors, one local recurrence, 2 lymph node metastases, and 1 lung metastasis (Supplementary Table 4). Primary tumor locations were the parotid gland (n=9), submandibular gland (n=1), and small buccal glands (n=1); the primary tumor location was not known in the case of lung metastasis.

Chromosomal imbalances were found in 6 of 13 SGAC. 5 of these cases showed losses, chromosomal gains were seen in one case. In general, the number of imbalances was low, ranging from 1 to 3 events per case. Recurrent findings included losses of 1p and 18q (2 cases, each).

Previously reported cytogenetic analyses of few examples of SGAC have shown structural aberrations at chromosomes 1, 4, 5, and 6 [2-4]. Furthermore, LOH was detected at 4p, 5q, 6p, 17p (40-52%), and less frequently at 1p, 1q, 4q, 5p, and 6q [5].

Supplemental References

1. Ellis G, Simpson RHW (2005) Acinic cell carcinoma. In: Barnes L, Eveson JW, Reichart P, Sidransky D (eds) World health organization classification of tumours. Head and neck tumours. IARC Press, Lyon, pp 216-218

2. Mark HF, Hanna I, Gnepp DR (1996) Cytogenetic analysis of salivary gland type tumors. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 82 (2):187-192

3. Sandros J, Stenman G, Mark J (1990) Cytogenetic and molecular observations in human and experimental salivary gland tumors. Cancer GenetCytogenet 44 (2):153-167

4. Stenman G, Sandros J, Dahlenfors R, Juberg-Ode M, Mark J (1986) 6q- and loss of the Y chromosome--two common deviations in malignant human salivary gland tumors. CancerGenet Cytogenet 22 (4):283-293

5. el-Naggar AK, Abdul-Karim FW, Hurr K, Callender D, Luna MA, Batsakis JG (1998) Genetic alterations in acinic cell carcinoma of the parotid gland determined by microsatellite analysis. Cancer Genet Cytogen 102 (1):19-24

Supplemental Table 1: Primer sequences

Primer / Sequence
K-ras 1F / 5´-GTG TGA CAT GTT CTA ATA TAG TCA-3´
K-ras 1R / 5´-GAA TGG TCC TGC ACC AGT AA-3´
K-ras 2F / 5´-TCA AGT CCT TTG CCC ATT TT-3´
K-ras 2R / 5´-TGC ATG GCA TTA GCA AAG AC-3´
EGFR 18F / 5′-GCT GAG GTG ACC CTT GTC TC-3′
EGFR 18R / 5′-ACA GCT TGC AAG GAC TCT GG-3′
EGFR 19F / 5′-GCT GGT AAC ATC CAC CCA GA-3′
EGFR 19R / 5′-GAG AAA AGG TGG GCC TGA G-3′
EGFR 20F / 5′-CAT GTG CCC CTC CTT CTG-3′
EGFR 20R / 5′-GAT CCT GGC TCC TTA TCT CC-3′
EGFR 21F / 5′-CCT CAC AGC AGG GTC TTC TC-3′
EGFR 21R / 5′-CCT GGT GTC AGG AAA ATGCT-3′

Supplemental Table 2. Molecular and Immunohistochemical Results in PACs

Case / Diagnosis / DCC / MYC / MSI / MLH1 / MSH2 / MSH6
01 / PAC / reduced / n.i. / n.i. / n.i. / n.i. / +
02 / PAC / normal / n.i. / MSS / + / + / n.i.
03 / PAC / normal / 2 / MSS / n.i. / + / +
04 / PAC / reduced / 2 / MSS / + / n.i. / n.i.
05 / PAC / n.i. / 2-3 / MSS / n.i. / + / +
06 / PAC / loss / 2 / MSS / + / + / +
07 / PAC / loss / 2 / MSS / + / + / +
08 / PAC / reduced / 2-3 / MSS / + / + / +
09 / PAC / normal / 3-5 / MSS / + / + / +
10 / PAC / reduced / 2-3 / MSI / + / + / n.i.
11 / PAC / loss / n.i. / MSS / n.i. / n.i. / n.i.
12 / PAC / normal / 2 / MSS / + / n.i. / (+)
13 / PAC / normal / 4-6 / MSS / + / + / +
14 / PAC / reduced / 4-6 / MSS / + / + / +
15 / PAC / reduced / n.i. / MSS / + / + / n.i.
16 / PAC / normal / 2 / MSS / n.i. / n.i. / +
17 / PAC / normal / 2 / MSS / + / + / +
18 / PAC / loss / 1-2 / MSS / n.i. / + / n.i.
19 / PAC / reduced / 1-3 / MSS / + / + / +
20 / PAC / loss / 4-6 / MSS / + / + / +
21 / PAC / reduced / n.i. / MSS / + / + / +
22 / PAC / reduced / n.i. / MSS / + / + / +
23 / PAC / reduced / 2 / MSS / + / + / +
24 / PAC / loss / 2 / MSS / + / + / +
25 / PAC / loss / 2 / MSS / + / + / +
26 / PAC / reduced / 6-10 / MSS / + / + / +
27 / PAC / reduced / 2 / n.i. / + / + / +
28 / Cystic PAC / loss / 2 / MSS / + / + / +
29 / Cystic PAC / loss / 2-3 / MSS / + / + / +
30 / MACNEC / loss / n.i. / MSS / + / n.i. / +
31 / MACNEC / loss / 2-3 / MSS / + / + / +
32 / MACNEC / reduced / 3-4 / MSS / n.i. / n.i. / n.i.
33 / MACNEC / reduced / 2 / MSS / + / + / +
34 / MACNEC / reduced / 2-3 / MSS / + / + / +
35 / MACNEC / normal / n.i. / MSS / + / + / +
36 / MACNEC / loss / 1-2 / n.i. / + / + / +
37 / MACNEC / reduction / 2-3 / MSS / + / + / +
38 / MACNEC / loss / 3-4 / n.i. / + / + / +
39 / PAC / reduced / 2 / n.i. / + / n.i. / n.i.
40 / PAC / reduced / 2 / MSI / + / + / +
41 / PAC / normal / 2-3 / MSS / + / + / +
42 / PAC / reduced / n.i. / MSS / + / + / +
43 / PAC / normal / n.i. / MSS / + / + / n.i.
44 / PAC / loss / n.i. / n.i. / + / + / n.i.
45 / PAC / reduced / 3-4 / n.i. / + / n.i. / n.i.
46 / PAC / reduced / n.i. / n.i. / + / + / n.i.
47 / PAC / normal / n.i. / MSS / + / n.i. / n.i.
48 / PAC / loss / 2 / n.i. / + / + / n.i.
49 / PAC / reduced / 2 / MSS / + / n.i. / +
50 / PAC / reduced / n.i. / MSS / + / + / +
51 / PAC / loss / n.i. / n.i. / + / + / +
52 / PAC / reduced / 4-6 / n.i. / + / + / +
53 / PAC / normal / 2 / MSS / + / + / +
54 / PAC / reduced / 3-4 / n.i. / + / + / +
55 / PAC / reduced / 4-6 / n.i. / + / + / +
56 / PAC / reduced / 1-2 / n.i. / + / + / n.i.
57 / PAC / loss / n.i. / n.i. / + / + / +

n.i., not informative; MSS, microsatellite stable; MSI, microsatellite unstable (high level); +, positive, ++strongly positive, -, negative, +/- focally positive, -/+ positive in only few cells; () weak staining intensity

Supplemental Table 3. Putative Therapeutic Markers in PACs

Case / Diagnosis / EGFR IH / EGFR / k-ras (exons 1, 2) / Braf V600E / Her2 / MGMT / L1CAM / HSP90
01 / PAC / n.i. / n.i. / n.i. / n.i. / n.i. / n.i. / n.i. / n.i.
02 / PAC / 80% / wta / 13 ggc >gac / - / 0 / - / - / +
03 / PAC / 90% / n.i. / n.i. / n.i. / 0 / + / - / (+)
04 / PAC / 100% / wta / Wt / n.i. / 0 / + / - / +
05 / PAC / n.i. / wta / Wt / - / n.i. / n.i. / + / n.i.
06 / PAC / 0 / wt / Wt / n.i. / 0 / + / + / +
07 / PAC / 0 / wta / Wt / - / 0 / + / (+) / +
08 / PAC / n.i. / wta / Wt / - / 0 / + / + / +
09 / PAC / n.i. / wt / Wt / - / 0 / + / (+) / +
10 / PAC / n.i. / wt / Wt / n.i. / 0 / n.i. / n.i. / n.i.
11 / PAC / n.i. / wt / Wt / n.i. / n.i. / n.i. / n.i. / n.i.
12 / PAC / 80% / wt / Wt / - / 0 / - / - / +
13 / PAC / 80% / wta / Wt / - / 0 / + / - / +
14 / PAC / n.i. / wt / Wt / - / 0 / + / (+) / +
15 / PAC / 0 / wta / n.i. / n.i. / 0 / - / (+) / +
16 / PAC / n.i. / wt / Wt / n.i. / n.i. / n.i. / ++ / n.i.
17 / PAC / 0 / wta / Wt / - / 0 / +- / (+) / +
18 / PAC / 0 / wt / Wt / n.i. / 0 / -+ / - / +
19 / PAC / 0 / wt / Wt / - / 0 / + / (+) / +
20 / PAC / 0 / wta / Wt / - / 0 / + / + / +
21 / PAC / 5% / wt / Wt / - / 0 / + / + / +
22 / PAC / 0 / n.i. / n.i. / n.i. / 0 / + / (+) / (+)b
23 / PAC / 0 / wta / Wt / - / 0 / - / (+) / +b
24 / PAC / 40% / wt / Wt / - / 0 / + / - / (+)
25 / PAC / 0 / wt / wt / - / 0 / - / (+) / (+)
26 / PAC / 5% / wt / wt / - / 0 / + / - / +
27 / PAC / 100% / wt / wt / - / 0 / + / (+/-) / +
28 / Cystic PAC / n.i. / wta / wt / - / n.i. / + / ++ / n.i.
29 / Cystic PAC / 100% / wt / wt / - / 0 / + / (+) / +
30 / MACNEC / n.i. / wta / wt / - / 0 / - / - / - b
31 / MACNEC / 0 / wt / wt / - / 0 / + / + / +
32 / MACNEC / n.i. / wt / wt / - / 0 / - / - / +
33 / MACNEC / 0 / wt / wt / - / 0 / +- / ++ / +/-
34 / MACNEC / 5% / wt / wt / - / 0 / -+ / + / +
35 / MACNEC / 0 / wt / wt / - / 0 / + / (+/-) / (+)/-
36 / MACNEC / n.i. / n.i. / n.i. / - / n.i. / n.i. / + / +
37 / MACNEC / 0 / wt / wt / n.i. / 0 / + / (+) / (+)b
38 / MACNEC / 100% / n.i. / n.i. / - / 0 / + / (+) / +
39 / PAC / 100% / wt / wt / n.i. / 0 / - / - / (+)
40 / PAC / 100% / wt / wt / - / 0 / +- / + / +
41 / PAC / 90% / wt / wt / - / 0 / + / (+) / +
42 / PAC / 5% / wt / wt / - / 0 / +- / (+/-) / + b
43 / PAC / 0 / wta / wt / - / 0 / - / - / +
44 / PAC / 0 / wta / wt / n.i. / 0 / - / - / + b
45 / PAC / 100% / n.i / n.i. / - / 0 / - / (+) / +
46 / PAC / 0 / n.i / n.i. / - / 0 / + / - / n.i.
47 / PAC / 0 / wta / n.i. / n.i. / 0 / - / (+) / +
48 / PAC / 0 / n.i. / n.i. / - / 0 / - / (+) / n.i.
49 / PAC / 0 / wta / wt / - / 0 / + / (+) / n.i.
50 / PAC / 90% / wta / 12 ggt > gat / - / 0 / + / (+/-) / +
51 / PAC / 0 / n.i / n.i. / - / 0 / + / (+) / +
52 / PAC / 0 / n.i. / n.i. / - / 0 / + / + / +
53 / PAC / 0 / wt / wt / - / 0 / + / - / +
54 / PAC / 0 / n.i. / n.i. / - / 0 / + / (+) / +
55 / PAC / 0 / n.i. / n.i. / - / 0 / + / + / +
56 / PAC / 90% / n.i. / n.i. / - / 0 / + / (+) / +
57 / PAC / 0 / n.i. / n.i. / n.i. / 0 / + / ++ / +

a, in these cases, not all exons were informative; b, nuclear staining; n.i., not informative; +, positive, ++strongly positive, -, negative, +/- focally positive, -/+ positive in only few cells; () weak staining intensity

Supplemental Table 4. Clinical, pathological and cytogenetic findings in 13 acinar cell carcinomas of salivary glands

Case / Material / Sex/ Age [years] / Primary location / Size [cm]1 / Ki-67 [%] / Gains / Losses
1 / LN / F/ 61 / Parotid gland / - / 20 / 12q14-15
20q13.1-13.3
2 / P / M/ 27 / Parotid gland / 3 / 10
3 / P / M/ 65 / Submandibular gland / 2 / 20
3 / LN / M/65 / Submandibular gland / - / 20 / 1p13-33
4q31.1-31.3
18q21-22
4 / P / F/ 77 / Parotid gland / 4 / 10 / 1p21-3
11q22-25
15q
5 / P / M/ 55 / Parotid gland / 3 / 15 / 18q12-22
6 / P / F/ 68 / Small buccal glands / n.i. / n.i. / 6q11-22
7 / P / M/ 32 / Parotid gland / 7.5 / 5
8 / P / F/ 64 / Parotid gland / 1.3 / 1
9 / P / F/ 32 / Parotid gland / n.i. / 5
10 / P / F/ 62 / Parotid gland / 6 / 5
11 / Lung M / M/ 67 / n.i. / - / 5 / 12p12-13
12 / REC / M/ 64 / Parotid gland / 4 / 5

1not indicated for metastases; P, primary tumor; REC, local recurrence; LN, lymph node metastasis, Lung M, lung metastasis

1