The Veterinary Journal 185 (2010) 62–67

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The Veterinary Journal

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Evaluation of clinico-pathological criteria and the Ki67 index as prognosticindicators in canine insulinoma

Floryne O. Buishand a, Marja Kik b, Jolle Kirpensteijn a,*

a Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, PO Box 80154, 3584 TD Utrecht, The Netherlandsb Department of Pathobiology, Division Pathology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands

a r t i c l e i n f o

Keywords:DogInsulinImmunohistochemistryPancreasBeta-cell tumourBiomarkers

1090-0233/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.tvjl.2010.04.015

* Corresponding author. Tel./fax: +31 30 2531776.E-mail address: j.kirpensteijn@uu.nl (J. Kirpensteij

a b s t r a c t

The establishment of reliable prognostic biomarkers for canine insulinoma are warranted to facilitateoptimal patient management. Using univariate and multivariate analyses, the present study evaluatedthe prognostic power of several clinical and histopathological criteria, as well as the Ki67 index, in 26dogs with insulinoma. On univariate analysis, stromal fibrosis within these tumours was found to be sig-nificantly predictive for survival, while tumour size, the Ki67 index, and TNM (Tumour, Node, Metastasis)stage were significant in the prognosis of both disease-free interval and survival time. On multivariateanalysis, tumour size retained predictive power for disease-free intervals, while the Ki67 index provedprognostically significant for both the disease-free interval and overall survival time. This study demon-strates that, in addition to known factors such as tumour size and stage, Ki67 can act as a biomarker ofinsulinoma that can be used to predict clinical outcome.

� 2010 Elsevier Ltd. All rights reserved.

Introduction

Insulinomas (INS) are pancreatic b-cell tumours characterisedby inappropriate secretion of insulin, leading to clinical signs asso-ciated with hypoglycaemia. There is confusion in the literatureconcerning their biological nature in the dog. Capen and Martin(1969) demonstrated that, based on histological and electronmicroscopic evaluation, 60% of INS were carcinomas and 40% wereadenomas. In contrast, Mehlhaff et al. (1985) found that the INSfound in 35 dogs were all adenocarcinomas. Currently, canineINS are assumed to be malignant in >95% of cases because,although they may lack the typical histological features of malig-nancy, they almost always metastasise (Leifer et al., 1986; Cay-wood et al., 1988).

Surgery is the treatment of choice for canine INS, as it prolongssurvival time (ST) (Tobin et al., 1999). This involves removal of theprimary tumour by partial pancreatectomy and the potentialremoval of metastases from lymph nodes or the liver. Where hypo-glycaemia recurs after surgery, dogs may benefit from treatmentwith glucocorticoids or diazoxide to prolong survival. The disease-free interval (DFI) and ST after partial pancreatectomy are difficultto predict because they have such a wide range. The median DFIof 31 dogs that underwent partial pancreatectomy at UtrechtUniversity between 1982 and 1997 was 244 days (range0–1116 days) and the median ST was 258 days (range 1–1146 days)

ll rights reserved.

n).

(Tryfonidou et al., 1998). In a recent study of this neoplasm, largevariations in DFI and ST were reported by Polton et al. (2007). Of19 dogs that underwent partial pancreatectomy, the median DFIwas 496 days (95% confidence interval [CI], 302–690 days) and themedian ST 785 days (95% CI, 190–1380 days).

Established immunohistochemical biomarkers of both humanand canine INS include insulin, chromogranin A, neuron-specificenolase, and synaptophysin (Myers et al., 1997; Kiupel et al.,2000; Madarame et al., 2009). However, the considerable variabil-ity in the behaviour of this tumour in the dog means that addi-tional prognostic biomarkers are required to optimise patientmanagement. The few prognostic factors currently available in-clude age, TNM (Tumour, Node, Metastasis) classification system,pre-operative serum insulin concentrations, and post-operativeblood glucose levels (Caywood et al., 1988; Tryfonidou et al.,1998). In humans, a number of histological parameters, includingmitotic index, nuclear atypia, tissue necrosis, histological structure,stromal fibrosis within the neoplasm, infiltrative growth patternand Ki67 index have shown prognostic significance (La Rosaet al., 2009). The objective of this current study was to assess theprognostic utility of a range of clinico-pathological and morpholog-ical criteria in the evaluation of canine INS.

Materials and methods

Case selection

Twenty-six canine INS from the archives of the Pathology Division of the Facultyof Veterinary Medicine, Utrecht University from 1993 to 2009, were selected. TheINS had been removed by partial pancreatectomy, using either the suture–fracture

F.O. Buishand et al. / The Veterinary Journal 185 (2010) 62–67 63

technique (n = 18) or the LigaSure Vessel Sealing System (n = 8) (Buishand andKirpensteijn, 2008). For each case, information on age, breed, gender, bodyweight,tumour size, location within the pancreas, presence of macroscopically-visiblemetastases at the time of surgery, and follow-up information on DFI and ST was col-lated. The possible presence of metastases was assessed both pre-operatively usingultrasound and computed tomography, as well as intra-operatively by carefulinspection and palpation of the abdominal viscera. For TNM staging, the criteriawere adapted from Rindi et al. (2006) (Table 1). Relevant clinico-pathological dataof the dogs are detailed in Table 2.

Histopathological examination

Twenty-three of the 26 cases were examined as the original slides for theremaining three cases could not be reviewed. Tissues were fixed in 10% neutral-buf-fered formalin for 24–36 h. After routine processing and paraffin embedding, sec-tions 5 lm thick were cut and stained with haematoxylin and eosin. Thefollowing morphological parameters were evaluated as described by La Rosa et al.(2009): tumour cellularity and architecture; growth pattern (expansile or infiltra-

Table 1Details of the TNM (Tumour, Node, Metastasis) classification system for canineinsulinoma.

T – primary tumourT0 – no evidence of tumourT1 – tumour in pancreas (size < 2 cm)T2 – tumour in pancreas (size > 2 cm)

N – regional lymph nodesN0 – not involvedN1 – involved

M – distant metastasisM0 – noneM1 – present

Clinical stagingStage I T1 N0 M0Stage II T2 N0 M0Stage III Any T N1 M0Stage IV Any T Any N M1

Table 2Clinico-pathological details of dogs with insulinoma (INS).

Case number Breed Sex Age (years) Weight (kg) Panclocal

1 Anatolian shepherd dog M 6 62.5 Lefta

2 Irish setter Mx 7 36.0 Left3 Bouvier des Flandres Fx 5 38.0 Righ4 Kooiker dog F 8 10.7 Righ5 Collie F 8 29.9 Righ6 Beagle Fx 5 14.8 Left7 German pointer M 9 31.4 Body8 Boxer M 6 30.0 Righ9 West highland white terrier M 8 10.0 Righ10 Labrador retriever M 10 53.0 Righ11 Belgian shepherd dog M 9 40.0 Left12 Boxer Fx 9 36.9 Left13 Belgian shepherd dog M 10 54.0 Left14 Boxer Fx 10 30.8 Left15 Bearded Collie Fx 8 22.5 Left16 Jack Russell terrier Fx 9 6.4 Left17 Crossbred M 10 20.0 Left18 Crossbred M 5 35.0 Left19 West highland white terrier F 7 7.4 Left20 Crossbred Fx 9 15.0 Left21 Bearded Collie Mx 10 22.0 Righ22 German shepherd dog F U 28.0 Left23 Maltese terrier M 9 5.5 Righ24 Yorkshire terrier Mx 6 3.7 Left25 Border Collie F 6 19.3 Righ26 Dachshund Fx 7 9.8 Body

Mx, castrated male; Fx, neutered female; U, unknown; AW, alive and without disease; DOfrom surgery until recurrence of clinical signs associated with INS; ST, time from surger

a Two primary tumours present.

tive); vascular or neural invasion; presence of necrosis and stromal fibrosis; nuclearatypia and mitotic index. Furthermore, tumours were given a score based on theirtendency to infiltrate (infiltrative pattern), a cumulative parameter representing acombination of at least two of the following: vascular invasion, infiltrative growthand fibrosis within the neoplasm.

Nuclear atypia was scored as: absent (regular, centrally-located and similar tothose of normal b-cells); mild (moderate increase in nuclear:cytoplasmic ratioand/or mild pleomorphism); moderate (large and pleomorphic with coarsely gran-ular or dispersed chromatin, sometimes with clearly visible nucleoli), or severe(irregularly-shaped hyperchromatic nuclei – small, intermediate, round or fusi-form). Tumour cellularity was scored as: ‘0’ (distribution of cells similar to that innormal islets); ‘1’ (distribution of cells similar to score 0, but in a few areas the nu-clei tend to approach each other); ‘2’ (significant increase in cell crowding, with aback-to-back pattern), or ‘3’ (densely packed cells with scant cytoplasm, nuclear‘moulding’ evident).

To assess the mitotic index, the slides were initially given a score at low mag-nification (40�) to identify areas with the highest number of mitotic figures. There-after, slides were examined under high magnification (400�), and the number ofmitoses in 40 high-power fields (HPF) was counted. These figures were then dividedby four to obtain the mitotic index/10 HPF. The mitotic index was scored as: ‘0’ (0–1mitotic figure/10 HPF); ‘1’ (2–10 mitotic figures/10 HPF), or ‘2’ (>10 mitotic figures/10 HPF). The histopathological scoring was performed by a board-certified morpho-logical pathologist (MK), without prior knowledge of the case details.

Immunohistochemical examination

To evaluate the Ki67 proliferative index, immunohistochemical staining of thetwenty-three INS was performed using rabbit monoclonal antibodies directedagainst Ki67 (Labvision RM-9106-S0, clone SP6). After paraffin removal and an anti-gen retrieval step, sections were treated with 0.3% H2O2 in methanol for 30 min toblock endogenous peroxidase activity. Slides were then rinsed in phosphatebuffered saline (PBS)/Tween-20 and ‘blocked’ for 30 min with 10% normal goat ser-um. Sections were incubated overnight at 4 �C with the primary antibody (dilution1:50). After this incubation, the slides were rinsed with PBS/Tween-20 and incu-bated for 45 min with Envision anti-rabbit (Dakocytomation K4003).

The slides were again rinsed in PBS and any tissue reaction visualised by adding3,30-diaminobenzidine. Slides were subsequently counter-stained with haematoxy-lin. The Ki67 index was determined by counting the fraction of positive tumourcells/2000 cells in areas with the highest degree of immuno-labelling. A tumour cellwas considered positive if its nucleus labelled positively for Ki67 (Fig. 1). Similarlyto La Rosa et al. (2009), Ki67 expression was categorised using a cut-off value of2.5% so that 62.5% was considered negative.

reaticisation

Diameter(mm)

Stage Outcome DFI (days) ST (days)

90 IV DOD 7 6940 II DOD 31 405

t 6 III DOD 0 48t 15 IV DOD 63 442t 40 IV DOD 122 166

15 I DOD 161 28627 II DOD 260 777

t 15 I DOD 302 462t 15 I DOD 343 344t 40 III DOD 462 464

20 II AW 247 24725 III DOD 509 64515 III DOD 510 623

and body 35 II DOD 472 56115 III AWD 546 104535 II DOUC 1042 104210 I AW 1154 115420 II AW 905 90512 I AW 819 819

8 III AW 1565 1565t 25 IV DOD 438 647

50 IV DOD 0 0t 10 III DOD 0 1

10 I AW 257 257t 5 I DOD 41 190

13 I AW 183 183

D, death due to INS; AWD, alive with INS; DOUC, dead of unknown cause; DFI, timey until either death or the end of the follow-up period.

Fig. 1. Photomicrograph illustrating Ki67 immuno-labelling of canine insulinomas:(A) positive staining within the nuclei of a subset of tumour cells (case number 12);(B) no staining observed (case number 13). Haematoxylin counter-stain.

Table 3Details of histopathological scoring and of the immunohistochemical determination of the

Casenumber

Nuclearatypia

Tumourcellularity

Tumourarchitecture

Necrosis Growthtype

1 Mild 1 Trabecular � Expanding2 Mild 1 Tubuloacinar � Infiltrative3 Mild 1 Trabecular � Expanding4 Mild 1 Solid nest + Infiltrative5 Moderate 1 Tubuloacinar + Infiltrative6 Severe 2 Solid nest + Infiltrative7 Moderate 2 Solid nest + Infiltrative9 Moderate 2 Undifferentiated + Infiltrative

10 Severe 2 Undifferentiated + Infiltrative12 Moderate 1 Tubuloacinar + Infiltrative13 Moderate 1 Solid nest � Infiltrative14 Moderate 2 Solid nest � Infiltrative15 Moderate 2 Solid nest � Expanding16 Severe 2 Solid nest + Infiltrative17 Moderate 2 Tubuloacinar � Infiltrative19 Moderate 2 Solid nest � Infiltrative20 Moderate 1 Tubuloacinar � Infiltrative21 Moderate 2 Solid nest � Expanding22 Mild 1 Tubuloacinar � Infiltrative23 Moderate 2 Solid nest + Infiltrative24 Moderate 2 Solid nest � Infiltrative25 Moderate 1 Solid nest � Infiltrative26 Absent 0 Solid nest � Infiltrative

+, Present; �, absent.a Ki67 expression categorised using a cut-off value of 2.5% so that 62.5% was conside

64 F.O. Buishand et al. / The Veterinary Journal 185 (2010) 62–67

Statistical analysis

With respect to DFI and ST, the predictive value of the clinico-pathologi-cal, histopathological, and immunohistochemical parameters were assessedusing univariate Cox proportional hazard regression models. Dogs with DFIof <7 days were excluded from the analysis. ST was excluded for those pa-tients still alive at the end of the monitoring period, or that died of othercause. Chi-square analysis was used to determine significance differences withtwo-sided P values of <0.05. Parameters with a P-value <0.15 on univariateanalysis were then subjected to multivariate analysis using the Cox propor-tional hazard regression model. The Kaplan–Meier product limit method wasused to assess the prognostic significance of the Ki67 index. The Mantel–Cox Log Rank test was used to determine the significance of the Kaplan–Me-ier product limit method. The SPSS 16 (SPSS Inc.) program was used for sta-tistical analyses.

Results

Clinico-pathological criteria

In 14/26 cases investigated, the tumour was confined to thepancreas. Eight tumours were <2 cm in size (stage I) and six wereP2 cm (stage II). In seven cases the INS had metastasised to locallymph nodes only (stage III) whereas in five cases both lymphnodes and the liver was involved (stage IV). TNM stages I–III dogshad significantly longer DFIs (P = 0.008; odds ratio [OR] 5.7; 95% CIfor OR: 1.6–20.8) and STs (P = 0.034; OR 3.2; 95% CI for OR: 1.1–9.2)compared to dogs with stage IV TNM. There were no significant dif-ferences between TNM stages I–III. Besides clinical stage, univari-ate analysis indicated that tumour size was statisticallysignificant in relation to DFI (P = 0.010; OR = 1.7; 95% CI for OR:1.1–2.4) and ST (P = 0.037; OR = 1.3; 95% CI for OR: 1.0–1.8).

Histopathological and immunohistochemical criteria

The results of histopathological scoring and of the immunohis-tochemical determination of the Ki67 index are outlined in Table 3.Univariate analysis found that the stromal fibrosis within the neo-plasm was a significant prognostic indicator for ST (P = 0.021; OR

Ki67 index in 23 canine insulinomas.

Stromalfibrosis

Vascularinvasion

Neuralinvasion

Infiltrativepattern

Mitoticindex

Ki67indexa

� + � � 0 >2.5� � � � 0 >2.5+ � � � 0 >2.5� + � + 0 >2.5+ + � + 1 >2.5� + � + 1 62.5� + � + 0 62.5� + + + 0 62.5� + � + 1 62.5� + � + 0 >2.5� + � + 0 62.5� � � � 1 62.5� � � � 1 62.5� + � + 0 62.5� + � + 1 62.5� + � + 0 62.5� + � + 0 62.5� � � � 1 >2.5� + � + 1 62.5� + � + 0 >2.5� + � + 1 >2.5� + � + 1 >2.5� + � + 2 >2.5

red negative (La Rosa et al., 2009).

F.O. Buishand et al. / The Veterinary Journal 185 (2010) 62–67 65

8.4; 95% CI for OR: 1.4–51.5). The other histopathological criteriaexamined did not have prognostic significance with regard toeither DFI or ST. Cases with a Ki67 index >2.5% had significantlyshorter DFIs (P = 0.014; OR 4.5; 95% CI for OR: 1.4–15.1) and STs(P = 0.022; OR 3.5; 95% CI for OR: 1.2–10.1), compared to thosewith a Ki67 index 62.5% (Fig. 2).

Multivariate analysis

On multivariate analysis of clinical staging both tumour size(P = 0.022; OR 1.6; 95% CI for OR: 1.1–2.5) and a Ki67 index of>2.5% (P = 0.023; OR 4.1; 95% CI for OR: 1.2–13.9) had predictivepower with regard to DFI. Multivariate analysis of clinical staging,tumour size, Ki67 index >2.5%, fibrosis within the neoplasm andtumour cellularity, indicated that the Ki67 index (P = 0.022; OR3.5; 95% CI for OR: 1.2–10.1) had predictive power with regard toST.

Fig. 2. Kaplan Meier disease-free (A) and survival (B) curves for dogs withinsulinoma with (dotted line) and without (solid line) Ki67 immuno-labelling.Censored cases are indicated with a ‘+’. There is a significant difference in disease-free interval (P < 0.01) and survival time (P = 0.016) between positive and negativecases as defined immunohistochemically.

Discussion

In this study, univariate analyses found that stromal fibrosiswithin canine INS is a useful prognostic indicator of ST, while theKi67 index, tumour size and TNM stage were useful in predictingboth DFI and ST. The tumour size and Ki67 index retained predic-tive power for DFI in a multivariate model, as did the Ki67 index forST.

There is some controversy regarding the relationship betweenthe histopathological features and the benign or malignant nat-ure of canine b-cell tumours (Capen and Martin, 1969; Mehlhaffet al., 1985; Leifer et al., 1986; Caywood et al., 1988), and clas-sifying these neoplasms, in both dogs and humans, as adenomasor adenocarcinomas based on their morphology, frequently doesnot reflect their biological behaviour (Minkus et al., 1997; Aliet al., 2006). Mehlhaff et al. (1985) highlighted the fact thatthe DFI and ST are similar in dogs with INS classified as adeno-mas and adenocarcinomas. Although, based on histopathologicaland ultrastructural criteria, Capen and Martin (1969) concludedthat 40% and 60% of canine INS were benign and malignant,respectively, there was little clinical evidence to support thisfinding.

The present study demonstrated that 96% of canine INS arehighly cellular and exhibit a certain degree of nuclear atypia. In83% of cases, these INS had an infiltrative growth pattern and89% of these tumours, in turn, had a cumulative ‘infiltrative pat-tern’. Although these percentages are in line with the malignantbehaviour of canine INS, overall, this study supports previouswork, which has highlighted the general lack of prognostic signif-icance of histopathological criteria (Kruth et al., 1982; Mehlhaffet al., 1985; Caywood et al., 1988). Increased stromal fibrosiswithin tumours was the only significant morphological prognosticmarker identified in our study. This feature has been found previ-ously to significantly predict the DFI and ST in human patientswith INS (La Rosa et al., 2009). Since myofibroblasts are a keycomponent of this fibrous response in INS in humans (Kurodaet al., 2004), and given these cells can promote neoplastic pro-gression, this might explain the negative impact of this processon clinical outcome.

TNM stage has been shown to be a good prognostic marker incanine b-cell tumours (Caywood et al., 1988). Dogs with stage I dis-ease (T1N0M0) had significantly longer DFI than animals in stage II(T1N1M0) or III (T1N0M1 or T1N1M1). Furthermore, dogs withstage III disease had a significantly shorter ST than dogs at stageI or II. As would be anticipated, the current study confirmed thatdogs with distant metastases survive for a significantly shortertime than dogs where the neoplasm is restricted to the pancreas

and regional lymph nodes. In contrast to the study of Caywoodet al. (1988), we did not find a significant difference in the DFI be-tween dogs with INS confined to the pancreas and animals withmetastatic disease. Our study demonstrated a significant differencein TNM stage for DFI which was similar for ST. Disease in dogs withdistant metastases remained sub-clinical for a significantly shorterperiod after surgery than in dogs where the neoplasm was con-fined to the pancreas and draining lymph nodes.

The difference in prognostic significance of TNM stage with re-gard to DFI, between the two studies may be due to differences in

66 F.O. Buishand et al. / The Veterinary Journal 185 (2010) 62–67

numbers of cases studied or advances in surgical technique. Liga-Sure, for example, is a novel electro-thermal bipolar sealing de-vice that facilitates removal of metastatic lesions in lymphnodes, that may possibly reduce the prognostic difference be-tween stages I and II dogs as described by Caywood et al.(1988). The lack of prognostic significance of stage I INS with re-gard to DFI, could also be due to the fact that 6/7 dogs that werestill disease-free at the end of study follow-up, had tumours con-fined to the pancreas. As time progresses, it will be possible todetermine the definitive DFI for these dogs and it thus remainspossible that the T1N0M0 stage may yet turn out to be a positiveprognostic marker.

Caywood et al. (1988) also demonstrated that age was a factorwith prognostic significance in cases of INS with older dogs surviv-ing significantly longer than younger animals. However, a more re-cent study, like ours, reported that age was not significant(Tryfonidou et al., 1998). The current study found that tumour sizewas a significant prognostic indicator, a feature previously found inhuman cases of INS, where larger tumours resulted in a poorerclinical outcome (Hochwald et al., 2002; Schmitt et al., 2007; LaRosa et al., 2009). The consistent association between large tumoursize and metastatic risk might also account for the increased risk ofmicro-metastases and poorer prognosis for dogs with large b-cellneoplasms (Minn et al., 2007).

Ki67 is a proliferation marker, expressed during the activephases of the cell cycle and absent from resting cells (Scholzenand Gerdes, 2000). The Ki67 index has often been correlated withclinical outcome in human INS (Pelosi et al., 1996; La Rosa et al.,2007, 2009). Our study indicated that high Ki67 expression resultsin a poor prognosis in canine INS. To the authors’ knowledge, theonly previous report of Ki67 expression in canine INS (Minkuset al., 1997) found that although the Ki67 index was higher in tu-mours that had metastasised, this difference was not statisticallysignificant. However significance levels may not have beenreached, as stages I and II tumours were compared to those instages III and IV. Taking into account the results of the presentstudy, discriminating between stages III and IV neoplasms mayhave resulted in a different outcome. The prognosis for dogs withmetastases beyond the regional lymph nodes is significantlypoorer than for animals with regional disease only, and high Ki67expression predicts a significantly worse clinical outcome.

Whereas TNM stage and tumour size criteria can be easily usedpre-operatively to provide a prognosis for dogs with INS, the Ki67index, can only be determined post-operatively. However, recentwork demonstrated that Ki67 expression in endoscopic ultraso-nography-guided fine needle aspirate cytology (FNAC) samples ofhuman INS was similar to those in histopathological samples (Pianiet al., 2008). Further studies are required to determine if this cor-relation exists in the dog.

Conclusions

The clinical behaviour of canine INS is difficult to predict be-cause of the lack of reliable prognostic criteria. The present studyhas demonstrated that tumour size, TNM stage, stromal fibrosisand the Ki67 index, are useful parameters in this context. TheKi67 index and tumour size were found to be particularly powerfulprognostic biomarkers as these parameters independently influ-enced patient survival.

Conflict of interest statement

None of the authors of this paper has a financial or personalrelationship with other people or organisations that could inappro-priately influence or bias the content of the paper.

Acknowledgements

The authors gratefully acknowledge the technical assistance ofA.M. van Ederen. Furthermore, we are grateful to Dr. Hans S. Koo-istra and Daniel J. Compton for critically evaluating the manuscript.

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