Cyclin-dependent kinase (cdk6) and p16 in pancreatic endocrine neoplasms

Cyclin-dependent kinase (cdk6) and p16 in pancreatic endocrine neoplasms

Pathology (December 2004) 36(6), pp. 566–570 ANATOMICAL PATHOLOGY Cyclin-dependent kinase (cdk6) and p16 in pancreatic endocrine neoplasms TATSUO TO...

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Pathology (December 2004) 36(6), pp. 566–570

ANATOMICAL PATHOLOGY

Cyclin-dependent kinase (cdk6) and p16 in pancreatic endocrine neoplasms TATSUO TOMITA Department of Pathology, Texas Tech Medical Center, El Paso, Texas and University of Kansas Medical Center, Kansas City, Kansas, USA

Summary Aims: p16 and p27, the inhibitors of cyclin-dependent kinases, have been reportedly decreased in certain human tumours, including a few endocrine tumours. The current study used immunocytochemical staining to compare the staining intensity of cdk6 and its inhibitor, p16, in pancreatic endocrine neoplasms with normal pancreatic islets. Methods: Twenty-four primary pancreatic endocrine neoplasms, consisting of 12 insulinomas, one glucagomoma, three pancreatic polypeptide (PP)-omas, five gastgrinomas and three non-fuctioning tumours, were immunocytochemically studied for cdk6 and p16 compared with the adjacent non-neoplastic islets. Results: In the normal islets, cdk6 staining was strongly positive for islet cell nuclei and cytoplasms, whereas p16 was strongly positively stained for islet cell cytoplasms. Insulinomas, glucagonoma, PP-omas and non-functioning tumours were weakly stained for cdk6 and p16. Among five gastrinomas, three tumours were moderately stained and two tumours were more weakly stained for cdk6 and p16. Thus, tumour cells were weaker stained for cdk6 and p16 compared with the strong staining of normal islet cells. No distinct immunostaining difference was observed among five kinds of pancreatic endocrine neoplasms. Conclusions: The decreased immunocytochemical staining for cdk6 and p16 is consistently observed in five kinds of pancreatic endocrine neoplasms. This decreased cdk6 and p16 in pancreatic endocrine neoplasms may be a part of the cell cycle event in tumour transformation and progression, and the same process may involve other endocrine tumours. Key words: Cyclins, cyclin-dependent kinases, p16, pancreatic endocrine neoplasms. Received 18 August 2003, accepted 28 May 2004

INTRODUCTION Cyclins were first identified in marine intervertebrates (clams) as protease whose accumulation and degradation oscillate during the cell cycle.1 It is the sequential activation and destruction of cyclins which provide the primary means of cell cycle replication.2 At least nine cyclin-dependent kinases (cdks1–9) have been identified in mammalian cells, together with more than 16 mammalian cyclins identified so far.2,3 Each of the cyclin/cdk complexes has been implicated in regulating transcriptional elongation

through phosphorylation of the carboxyl-terminal domain of the largest subunit of RNA polymerase II.1 Passage through G1 into S phase is regulated by the action of cyclin D-, cyclin E- and cyclin A-associated kinases.4 The major cyclins regulating the G1 phase are cyclins D and E.5 The D-type cyclins and their associated kinases, cdk4 and cdk6, regulate phosphorylation and inactivation of the Rb protein.5,6 Two families of cdk inhibitors have been identified: the INK4 family7 and the CIP/KIP family.8 INK4 members p15, p16, p18 and p19 bind cdks, especially cdk4 and cdk6.7 While studying pancreatic exocrine neoplasms, islet cells were incidentally found to be immunocytochemically positive for cdk6 and p16.8 The current study was undertaken using immunocytochemical staining to identify cdk6 and its inhibitor p16 in pancreatic endocrine neoplasms.

MATERIALS AND METHODS A total of 24 primary pancreatic endocrine neoplasms were studied, obtained between 1974 and 2000 from the University of Kansas Medical Center, consisting of 12 insulinomas, one glucagonoma, five gastrinomas, three PP-omas, and three non-functioning endocrine neoplasms (Table 1). Twenty-three tumours were surgically removed and one tumour was collected at autopsy. All tissue specimens were routinely fixed in 10% neutral formalin and embedded in paraffin. Five normal pancreata were also included, consisting of three cases of patients with ductal adenocarcinoma and two cases of patients with benign cystadenoma. All deparaffinised sections were treated with 0.1 N citrate buffer (pH 6.0) at 100‡C for 10 min.9 For immunoperoxidase staining for all four pancreatic hormones, gastrin and chromogranin A (CGA), deparaffinised sections were treated with the antigen retrieval procedure and were processed as previously reported.10,11 For cdk6 and p16 immunocytochemical staining, deparaffinised sections were initially incubated with rabbit cdk6 and p16 antiserum (both from Santa Cruz Laboratory, USA) at 1:200 dilution overnight at 4‡C, followed by streptavidin-congugated goat anti-rabbit serum and subsequently with biotin-conjugated peroxidase. We defined the strongest staining intensity as 3z, moderate staining as 2z, weak staining as 1z and negative staining as 0.10,11

RESULTS Normal pancreatic islets The cytoplasms of normal pancreatic islet cells were moderately and diffusely positive for both cdk6 and p16 (Fig. 1). A stronger positive staining was noted for cdk6 in the nuclei and cytoplasms, whereas p16 staining was mostly in the cytoplasms (Fig. 1).

ISSN 0031-3025 printed/ISSN 1465–3931 # 2004 Royal College of Pathologists of Australasia DOI: 10.1080/00313020400011342

CDK AND P16 IN PANCREATIC ENDOCRINE NEOPLASMS

TABLE 1

Summary of case and results of cdk6 and p16 immunostaining

Neoplasm

Case no.

Age/sex

Location

Size (cm)

1 2 3 4 5 6 7 8 9 10 11 12

17/F 19/F 20/F 47/F 52/M 64/F 68/F 68/F 69/M 71/M 79/F 84/F

Head Body Tail Head Tail Tail Head Tail Body Head Head Body

1

43/F

Head/tail

1 2 3 4 5

29/F 29/F 44/F 47/F 54/M

Head Tail Duodenum Head Duodenum

1 2 3

26/F 33/M 74/F

Head Body/tail Head

1.561.0 15613614 1.061.0

1 2 3

42/F 58/M 66/M

Body/tail Body/tail Body

116668 86666 0.860.6

Insulinomas (n~12) 1.561.0 1.561.5 1.861.8 0.760.5 1.261.0 7.067.0 1.761.3 7.067.0 1.961.6 1.461.2 1.561.4 1.761.3

Glucagonoma (n~1) 1861068

Gastrinomas (n~5) 1.560.8 0.860.5 0.860.6 1.561.0 1.061.0

PP-omas (n~3)

Non-functioning (n~3)

Pancreatic endocrine neoplasms Among 12 primary insulinomas, insulin and CGA immunostaining was relatively weak for the tumours compared with the adjacent normal islets. The tumours were relatively weakly stained for cdk6 and p16 in 10 cases (Fig. 2; Table 1) and two cases were negative for both cdk6 and p16. Among five gastrinomas, three cases were moderately positive for both cdk6 and p16 (Fig. 3; Table 1) and the remaining two cases were weakly positive for both cdk6 and p16. Two gastrinomas, which were strongly stained for gastrin, were moderately positive for both cdk6 and p16, whereas two weakly stained tumours for gastrin were weakly stained for both cdk6 and p16 (Table 1). One glucagonoma, three PP-omas and three non-functioning

A

567

Insulin

CGA

cdk-6

p16

z zzz zz zz zz z zz zz z zzz zz zz Glucagon z Gastrin z z zzz zzz zz PP z z z Hormones 0 0 0

zz zz zz z zz z zzz zz z zzz zz zz

z 2 zz z z 2 z z z z z zz

z 2 z z z 2 z z zz 2 zz z

z

z

z

zz z zzz z z

z z zz zz zz

z z zz zz zz

zz z z

z z z

z z z

zz z zz

z z z

z z z

tumours were all weakly positive for both cdk6 and p16 (Fig. 4; Table 1). The positive staining of cdk6 and p16 in pancreatic endocrine neoplasms occupied mainly the tumour cell cytoplasms. Less positive staining of cdk6 and p16 was noted for insulinomas and more positive staining was observed for gastrinomas in this study (Table 1).

DISCUSSION The significance of cdk6 and p16 in pancreatic islets and pancreatic endocrine neoplasms is unknown at present as the finding of immunocytochemically positive staining for

B

Fig. 1 Normal pancreatic islet. The cytoplasms of normal islet cells are moderately and diffusely positive for cdk6 and p16 with scattered strong positive staining in the nuclei (*) and cytoplasms. (A) cdk6 immunostaining (original magnification, 6650). (B) p16 immunostaining (original magnification, 6650).

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A

B

Fig. 2 Insulinoma Case 10. The tumour cells are only weakly positive for cdk6 at the periphery of the tumour but are negative for p16, respectively. I, islet in the adjacent pancreas; T, tumour. (A) cdk6 immunostaining (original magnification, 6265). (B) p16 immunostaining (original magnification, 6265).

A

B

Fig. 3 Gastrinoma Case 5. The tumour cells are located in the submucosa and the tumour cells are moderately positive for both cdk6 and p16. M, mucosa; T, tumour. (A) cdk6 immunostaining (original magnification, 6650). (B) p16 immunostaining (original magnification, 6650).

cdk6 and p16 in pancreatic tissue was serendipitous. It appears that islet cells and other endocrine cells contain many more markers than chromogranins, synaptophysin, neuron-specific enolase and Leu-7.8 Islet cells in particular have been found positive for matrix metalloproteinases, tissue inhibitors of metalloproteinases,10 metallothionein,11 prohormone convertase 1/3 and 2,12 amylin13 and insulinlike growth factor.14 p16 is an INK4 family member and is an inhibitor of cdk6 via binding to the cdk6.15 Recently, immunocytochemical studies on p16 have been reported. Reithdorf et al. studied p16 immunocytochemical staining in endocervical glandular neoplasms and found p16-positive staining in cervical squamous cell carcinoma. They claimed a strong relationship between p16 expression and human papillomavirus (HPV)-encoded E6/E7 transfection.16 Baba et al. studied immunocytochemical staining of nasopharyngeal carcinoma and found reduced expression of p16 and p27 in the carcinomas compared with non-cancerous mucosa.17 In the last 3 years there has been a flood of publications on p27 for various cancers.18–26 So far, the published data appear to support the notion that decreased p27 immunocytochemical staining correlates with malignant

transformation in cancer tissues and also suggest poor prognosis.3,4,19–24,26 p27 immunocytochemical studies have been reported for some endocrine tumours. Erickson et al. studied p27 in Grave’s disease, in which papillary hyperplasia revealed 68% positive staining as compared with 26% positive staining for papillary carcinoma occurring in thyroid tissue of Grave’s disease.27 Nakabayashi et al. studied p27 immunocytochemical staining with non-recurrent and recurrent pituitary adenomas, and found p27 staining was 67% positive for non-recurrent tumours as compared with 47% positive staining for recurrent adenomas.28 These results also support decreased p27 immunostaining as a potential recurrence marker.28 At present, cdks, p27 and p16 appear to be the most promising markers for carcinomas and as prognostic indicators in many carcinomas.16,19,24 Decreased p16 and p27 have been implicated in the carcinogenesis of cervical adenocarcinoma and nasopharyngeal carcinoma.16,17 The positive staining for cdk6 and p16 in pancreatic endocrine neoplasms was mostly in the tumour cell cytoplasms. Similar cytoplasmic immunostaining was also reported for p53 in glioblastomas of the brain, in which cytoplasmic-positive staining was associated with

CDK AND P16 IN PANCREATIC ENDOCRINE NEOPLASMS

A

B

C

D

569

Fig. 4 Non-functioning tumour Case 3. The tumour cells are negative for insulin but positive for CGA, whereas the tumour cells are only weakly positive for both cdk6 and p16. I, islet in the adjacent pancreas; T, tumour. (A) Insulin immunostaining (original magnification, 6265). (B) CGA immunostaining (original magnification, 6265). (C) cdk6 immunostaining (original magnification, 6265). (D) p16 immunostaining (original magnification, 6265).

one normal and one mutative copy of p53 in the tumour cells by a combined immunocytochemical staining and amino acid sequence study of p53.29 The current study showed less immunocytochemical staining of cdk6 and p16 for insulinomas and more immunostaining for gastrinomas, although no distinct immunostaining difference was noted among five kinds of pancreatic endocrine neoplasms (Table 1). The significance of p16 and p27 in tumorigenesis and tumour progression in pancreatic endocrine neoplasms is unknown at present. This simple immunocytochemical staining provides crucial information about cell cycle disruption in human tumours, which may render endpoints of further therapeutic monitoring. Lilja et al. identified cdk5 in insulin secreting beta-islet cells and found that an inhibition of cdk5 reduced glucoseinduced insulin secretion.30 Recently, significantly increased beta-cell mass has been revealed in cdk4 knock-in mice, suggesting that cdks are crucial for betacell development and insulin secretion.31 It is very likely that cdks and p16 are involved in insulin synthesis and secretion; furthermore, cdks and p16 may modulate synthesis and secretion of glucagon, somatostatin and PP either directly or indirectly. Address for correspondence: Dr T. Tomita, Department of Integrative Bioscience, Oregon Health and Science University, 611 SW Campus Drive, Portland, OR 97239-3097, USA. E-mail: [email protected]

References 1. Rosenthal ET, Hurst T, Ruderman JV. Selective translation of mRNA controls: The comparison of protein synthesis during early development of the surf clam. Cell 1980; 20: 489–94. 2. Johnson DG, Walker CL. Cyclins and cell cycle check-points. Annu Rev Pharmacol Toxicol 1999; 39: 295–312. 3. Tsihlias JJ, Kapusta L, Slingerland J. The prognostic significance of altered cyclin-dependent kinase inhibitors in human cancer. Annu Rev Med 1999; 50: 401–32. 4. Tsihlias J, Kapusta LR, DeBooer G, Morava-Protzner I, Zbieranowski I, Bhattacharya N. Loss of cyclin-dependent kinase inhibitor p27 is a novel prognostic factor in localized human prostate adenocarcinoma. Cancer Res 1998; 58: 542–8. 5. Sherr CJ. Cancer cell cycles. Science 1996; 274: 1672–7. 6. Sherr CJ. G1 phase progression: cycling on cue. Cell 1994; 79: 551–5. 7. Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ. Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase cdk4. Genes Dev 1993; 7: 331–42. 8. Tomita T. New markers for pancreatic islets and islet cell tumors. Pathol Int 2002; 52: 425–32. 9. Cattoretti G, Becker MH, Key G, et al. Monoclonal antibodies against recombinant parts of Ki-67 antigen detect proliferating cells in microwave-processed formalin-fixed, paraffin sections. J Pathol 1992; 168: 357–63. 10. Tomita T, Iwata K. Gelatinases and inhibitors of gelatinases in pancreatic islets and islet cell tumors. Mod Pathol 1997; 10: 47–54. 11. Tomita T. Metallothionein in pancreatic endocrine neoplasms. Mod Pathol 2000; 13: 389–95. 12. Tomita T. Immunocytotochemical localization of prohormone convertase 1/3 and 2 in pancreatic islets and islet cell tumors. Pancreas 2001; 23: 172–6. 13. Tomita T. Amylin in human pancreatic islets. Pathology 2003; 35: 34–6.

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14. Hoog A, Hu W, Abdel-Halim SM, Falkmer S. Ultrastructural localization of insulin-like growth factor-2 to the secretary granules of insulin cells. Ultrastruct Pathol 1997; 21: 457–66. 15. Alcorta DA, Xiong Y, Phelps D, Hannon C, Bearch D, Barrett C. Involvement of the cyclin-dependent kinase inhibitor p16 in replicative senescence of normal human fibroblasts. Proc Natl Acad Sci USA 1996; 93: 13742–7. 16. Reithdorf L, Reithdorf S, Lee KR, Cviko A, Loning T, Crum CP. Human papillomavirus expression of p16 and early endocervical glandular neoplasm. Hum Pathol 2002; 33: 899–904. 17. Baba Y, Tsukuda M, Mochimatsu I, Fukukawa S, Kagata H, Satake S. Reduced expression of p16 and p27 proteins in nasopharygeal carcinoma. Cancer Detect Prev 2001; 25: 414–9. 18. Kouvaraki M, Gorgoulis VS, Rassidakis GZ. High expression levels of p27 correlate with lymph node status in a subset of advanced invasive breast carcinomas. Cancer 2002; 94: 2454–65. 19. Vis AN, Noordzij MA, Fitoz K, Wildhagen MF, Schroder FH, van der Kwast TH. Prognostic value of cell cycle proteins p27kip1 and MIB-1, and the cell adhesion protein CD 44s in surgically treated patients with prostate cancer. J Urol 2000; 164: 2156–61. 20. Shibata H, Matubara O, Wakiyama H, Tanaka S. The role of cyclindependent kinase inhibitor p27 in squamous cell carcinoma of the esophagus. Pathol Res Pract 2001; 197: 157–64. 21. Thomas GU, Szugetu K, Murphy M, Dracetta G, Pagano M, Loda M. Down-regulation of p27 is associated with development of colorectal adenocarcinoma metastasis. Am J Pathol 1998; 153: 681–7. 22. Hashimoto K, Nio Y, Sumi S, et al. Correlation between TGF-a beta 1 and p21(WAF1/CIP1) expression and prognosis in respectable invasive ductal carcinoma of the pancreas. Pancreas 2001; 22: 341–7. 23. Tanmapfel A, Grund D, Katalinic A, et al. Decreased expression of

24.

25.

26.

27.

28.

29.

30.

31.

p27 protein is associated with advanced tumor stage in hepatocelluar carcinoma. Int J Cancer 2000; 89: 350–5. Huang LW, Chao SL, Hwang JL, Chou YY. Down-regulation of p27 is associated with malignant transformation and aggressive phenotype of cervical neoplasms. Gynecol Oncol 2002; 85: 524–8. Watanabe J, Sato H, Kanai T, et al. Paradoxical expression of cell cycle inhibitor p27 in endometrioid adenocarcinoma of the uterine corpus—Correlation with proliferation and clinicopathological parameters. Br J Cancer 2002; 87: 81–5. Shigemasa K, Shiroyama Y, Sawasaki T, et al. Underexpression of cyclin-dependent kinase inhibitor p27 is associated with poor prognosis in serous ovarian carcinomas. Int J Oncol 2001; 18: 953–8. Erickson LA, Yousef OM, Jin L, Lohse CM, Pankratz VS, Lloyd RV. p27kip1 expression distinguishes papillary hyperplasia in Grave’s disease from papillary thyroid carcinoma. Mod Pathol 2000; 13: 1014–9. Nakabayashi H, Sunada H, Hara M. Immmunohistochemical analysis of cell cycle-regulated protein, apoptosis and proliferation in pituitary adenomas. J Histochem Cytochem 2001; 49: 1193–4. Ali IU, Schweitzer JB, Ikejiri B, Saxena A, Robertson JT, Oldfield EH. Heterogeneity of subcellular localization of p53 pattern in human glioblastomas. Cancer Res 1994; 54: 1–5. Lilja L, Yang SN, Webb DL, Juntti-Berggren I, Bergren PO, Bark C. Cyclin-dependent kinase 5 promotes insulin exocytosis. J Biol Chem 2001; 276: 34199–205. Marzo N, Mora C, Fabregat ME, et al. Pancreatic islets from cyclindependent kinase 4/R24C (cdk4) knockin mice have significantly increased beta cell mass and are physiologically functional, indicating that cdk4 is a potential target for pancreatic beta cell mass regeneration in Type 1 diabetes. Diabetologia 2004; 47: 686–94.