The Apoptotic Paradox in Retinoblastoma

NATURAL COMPOUNDS AND THEIR ROLE IN APOPTOTIC CELL SIGNALING PATHWAYS

The Apoptotic Paradox in RetinoblastomaRita S. Sitorus,a,c Saukani Gumay,b and Paul van der Valkc

aDepartment of Ophthalmology, bDepartment of Pathology, Faculty of MedicineUniversity of Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia

cVrije University Medical Center, Department of Pathology, Amsterdam, the Netherlands

The purpose of this study was to investigate the clinicopathological features and the ex-pression of proteins involved in cell proliferation and the different pathways of apoptosisin retinoblastoma. Nineteen retinoblastoma patients were included, and mitotic index(MI) and apoptotic index (AI) were assessed. The expression of MIB-1, p53, caspase-3,Bcl-2, and Fas protein was assessed by immunohistochemistry. Mann–Whitney U testand Fisher’s exact test were used for statistical comparison. High MI (mean 16.84, range0–66) and high MIB-1 expression (mean 57.89, range 0–90) were found. The MI was signif-icantly related to MIB-1 expression (P = 0.01). The tumors showed a high apoptotic index(mean 40.26, range 1–110), and the AI was associated with the mitotic index (P = 0.02).The caspase-3 expression was positively related to the AI (P = 0.03), although a smallnumber of tumors with no significant or very low caspase-3 staining showed a highnumber of apoptotic cells, suggestive of a caspase-3-independent apoptosis pathway.Bcl-2 expression was not significantly related to AI (P = 0.07). No striking relationshipwas found in expression patterns of p53, Bcl-2, caspase-3, and Fas. In conclusion, wefound that (1) cell proliferation and apoptosis are linked in retinoblastoma; (2) activa-tion of effector caspase-3 induces apoptosis in retinoblastoma, but Bcl-2 overexpressiondoes not prevent apoptosis in many tumors; (3) there is a p53-independent pathway inapproximately one-quarter of cases; (4) the findings suggesting a caspase-3-independentpathway might lead to apoptosis in retinoblastoma; and, finally, we found no consis-tent pattern of expression of apoptotic and antiapoptotic molecules, suggesting that inretinoblastoma there is no preference for any single pathway of apoptosis. Confirmationof the results in a large set of tumors would be useful.

Key words: proliferation; apoptosis; mitotic index; apoptotic index; MIB-1; caspase;caspase-3; Fas receptor; p53; Bcl-2; retinoblastoma

Introduction

Retinoblastoma is the most common intraoc-ular malignancy in childhood. The retinoblas-toma susceptibility gene (RB), a prototypicaltumor suppressor, has been shown to be animportant regulator of cell cycle and apopto-sis. Tumors grow because the homeostatic con-trol mechanisms that maintain the appropriatenumber of cells in normal tissues are defec-

Address for correspondence: Rita S. Sitorus, M.D., Ph.D., Depart-ment of Ophthalmology Faculty of Medicine, University of Indonesia,Cipto Mangunkusumo Hospital, Salemba 6, Jakarta-10430, Indonesia.Voice/fax: +6221-3193-4878. [email protected]

tive, leading to an imbalance between cell pro-liferation and cell death. These two variableshave also been frequently quoted as havingprognostic significance in various human can-cers,1–8 and it is now widely accepted9–11 thatimpaired apoptosis not only plays a crucial rolein tumorigenesis but also facilitates metastasisand is a significant impediment to cytotoxictherapy.12–14

Chemotherapy-induced apoptosis is gener-ally thought to be dependent on activation ofcaspases. The caspase family of cysteine pro-teases is a central effector in apoptotic cell deathand is absolutely responsible for the morpho-logical features of apoptosis.15 Because the indi-cations for chemotherapy in the management

Natural Compounds and Their Role in Apoptotic Cell Signaling Pathways: Ann. N.Y. Acad. Sci. 1171: 77–86 (2009).doi: 10.1111/j.1749-6632.2009.04719.x c© 2009 New York Academy of Sciences.

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78 Annals of the New York Academy of Sciences

of retinoblastoma are increasing, study on thecaspase-mediated apoptotic pathway is be-coming more important. To date investiga-tions of the caspase-mediated apoptosis path-way in retinoblastoma are relatively limitedon the level of in vitro and experimentalstudies.16,17

Briefly, there are two separate pathways de-scribed,5,9,18 one activated by the death re-ceptor Fas and involving caspase-8; the other,the mitochondrial pathway, involving caspase-9 and the bax/bcl-2 family of proteins. Bothpathways converge on caspase-3, the commonend pathway and the ultimate effector of pro-grammed cell death.

The purpose of this study is to investigate theclinicopathological features of cell proliferationand apoptosis in retinoblastoma tumors and toevaluate, through a limited number of stain-ings, the involvement of the different apoptoticpathways.

Materials and Methods

Twenty-one enucleation or exenterationspecimens from 20 Indonesian patients withdiagnosis of RB were collected; two were ex-cluded because of completely necrotic tumors.Of the 19 sections from 19 patients selectedfor the study, 10 were derived from intraoculartumors, whereas nine were from extraoculartumors. Family history was negative in 18 pa-tients. None of the patients whose tumors wereexamined had received prior chemotherapyor radiotherapy before enucleation. We eval-uated hematoxylin and eosin (H&E)-stainedsections. Data tabulated included mitosis in-dex (MI), apoptotic index (AI), necrosis, cal-cification, grade of differentiation, unifocalor multifocal tumor, infiltration of the tu-mor in the optic nerve, choroid or sclera,and orbit. Data regarding gender, age, later-ality, family history, and clinical staging wererecorded.

Immunohistochemical Staining forMIB-1, p53, Bcl-2, and Caspase3

The paraffin-embedded tissues of 19 avail-able sections were retrieved for sectioning andimmunohistochemical staining. Deparaffinizedsections were immersed in methanol contain-ing 0.3% hydrogen peroxide for 30 min to blockendogenous peroxidase activity.

All slides for MIB-1 and p53 staining werepretreated with an antigen retrieval method byheating the slides in an autoclave (80◦C) in0.01 mol citrate buffer, pH 6.0, for 1 h. ForBcl-2 and caspase-3 staining, antigen retrievalwas applied by heating the slides up to 100◦Cfor 10 min and boiled at 360 W for 10 minin a microwave oven in Tris /EDTA or cit-rate buffer, respectively. Afterwards the slideswere incubated with the primary mouse mon-oclonal antibodies against p53 (DO-7; Dako,Glostrup, Denmark) in a 1:500 dilution; MIB-1 (AMAC Inc, Westbrok, ME) in a 1:40 di-lution; Bcl-2 (M0887; Dako) in a 1:150 di-lution; and caspase-3 (9661; Cell SignalingTechnology, Beverly, MA) in a 1:500 dilution.All slides were incubated overnight at 4◦Cexcept for Bcl-2, which was incubated 1 hat room temperature. Thereafter, slides wererinsed in PBS and incubated for 30 min at roomtemperature with sABC-HRP (streptavidin-biotin-conjugated horseradish peroxidase). Fi-nally, sections were washed in PBS and devel-oped with diaminobenzidine tetrahydrochlo-ride substrate (DAB; Chromogen, Carpinteria,CA). Sections were counterstained with hema-toxylin, dehydrated, and coverslipped.

Positive control tissues included tonsil (MIB-1, Bcl-2, caspase-3) and glioma (p53). Negativecontrols consisted of emission of the primaryantibody.

Activated caspase-3 (Asp 175) antibodyused in the present study detects endogenouslevels of the large fragment (17/19 kDa) of ac-tivated caspase-3 resulting from cleavage ad-jacent to Asp175. The antibody does not rec-ognize full-length caspase-3 or other cleavedcaspases.

Sitorus et al.: Apoptosis and Retinoblastoma 79

Immunohistochemical Stainingfor Fas/APO-1/CD95

For immunohistochemical staining of Fasprotein, a purified rabbit polyclonal antibodyagainst a peptide mapping at the carboxy ter-minus of FAS of human origin (C-20, sc-715;Santa Cruz Biotechnology, Santa Cruz, CA)was used.

Antigen retrieval was applied by heating thesections in a 0.01 mol citrate buffer (pH 6.0) in amicrowave at 100◦C for 15 min. Afterwards thesections were cooled at room temperature for atleast 30 min and washed with PBS. The slideswere preincubated 10 min with normal goatserum (1:10) at room temperature to diminishnonspecific binding of the secondary antibody.Slides were then incubated overnight at 4◦Cwith the primary antibodies against Fas in a1:400 dilution. Thereafter, slides were washedin PBS and incubated for 30 min at room tem-perature with sABC-HRP (streptavidin-biotin-conjugated horseradish peroxidase). Finally,sections were washed in PBS and developedwith DAB (Chromogen). Sections were coun-terstained with hematoxylin, dehydrated, andcoverslipped. The negative control was incu-bated with isotype-matched irrelevant antibod-ies. Positive control tissues used included lym-phoma and a hepatic adenocarcinoma.

Counting Mitoses

In the H&E staining, the most cellular areacontaining the highest density of mitotic figureswas selected. Mitotic figures were counted in 10consecutive high-power fields (HPF) at a 400×magnification, using a 40× objective/10× ocu-lar, starting at the spot within the measurementfield with the highest density of mitotic figures.The total number of mitotic figures counted inthese 10 fields was taken as the MI.

Counting of Apoptotic Cells

Apoptotic figures were counted using astandard light microscope of a 1000× mag-

nification (100× objective) in 10 fields ofvision. Everything from a dense, small, py-knotic nucleus in a deeply eosinophil cyto-plasm up to a group of dark nuclear rem-nants was considered as apoptotic cells. Thetotal number in 10 fields was taken as theAI.

Immunoscoring

Immunohistochemical stainings were scoredsemiquantitatively per high power field of400× (40× objective, 10× ocular, field diam-eter 450 μm) by two observers (P.vdV. andR.S.). Scoring was based on the percentageof tumor cells positively staining in the regionof tumor with the greatest density of stain-ing; the percentage of cells expressing pos-itive staining was analyzed as a categoricalvariable.

MIB-1-, p53-, and caspase-3-positive stain-ing were defined as the percentage of intactcells expressing nuclear staining; for Fas/Apo-1/CD95 the positivity was defined as the per-centage of cells expressing membrane or cyto-plasmic staining; and for Bcl-2 the percentageof intact cells expressing cytoplasmic staining.Staining for p53 and Bcl-2 was evaluated as theproportion of percentage of positive staining intwo categories (< 10%, ≥ 10%) and (< 50%,≥ 50%), respectively. The Fas receptor wasevaluated as positive and negative: for Fas neg-ative (< 10%) and positive (≥ 10%). Caspase-3was evaluated as low (1-< 5%) and high (≥ 5%)expression.

Statistical Analysis

Statistical analysis was performed usingSPSS software version 11.5 (SPSS, Inc.,Chicago, IL). Comparison of different groupswas performed with the Mann–Whitney U testor Fisher’s exact test, with P < 0.05 consideredsignificant.


TABLE 1. Histopathological Parameters and Staining Patterns of Cell Proliferation and Apoptosis inRetinoblastoma

No Tumor MI AI MIB-1 (%) p53 (%) Bcl-2 (%) Casp-3 (%) Fas

1 IO 23 39 60 0 0 10H +2 IO 4 80 90 0 0 5H −3 IO 4 8 20 10 0 1L +4 IO 14 20 25 25 30 1L +5 IO 14 40 50 40 50 2L +6 EO 0 3 50 1 5 <5L +7 IO 3 63 30 10 0 45H +8 IO 4 50 50 80 30 30H +9 EO 5 4 75 75 0 10H +10 IO 0 1 50 1 0 1L −11 EO 37 23 80 80 0 5H −12 EO 1 91 80 40 0 30H −13 EO 1 18 30 0 0 5H −14 EO 11 11 70 20 0 30H −15 EO 2 1 40 50 0 <5L −16 IO 66 65 80 70 40 10H +17 IO 50 73 80 70 80 10H +18 EO 30 110 80 80 80 20H +19 EO 51 66 60 10 5 1L +

MI, mitotic index; AI, apoptotic index; H, high; L, low; IO, intraocular; EO, extraocular.

Results

Clinical Data

A total of 19 sections of 19 patients wereincluded in this study. There were 14 boys andfive girls, with an age range of 7–66 (median 24)months. Six were bilateral cases and 13 wereunilateral. Ten tumors were located intraocular(IO) and nine had extended extraocular (EO)exhibiting obvious proptotic eyes clinically.

Cell Proliferation

Table 1 and Figure 1A–F summarize thehistopathological parameters and immunohis-tochemical pattern of cell proliferation-relatedproteins. MI ranged from 0 to 66 (mean 16.84),and MIB-1 expression ranged from 20–90%(mean 57.89) (Fig. 1C). High expression ofMIB-1 (> 50% positive staining) was found in58% of tumors. The MI was positively cor-related to MIB-1 expression (Mann–Whitney,P = 0.01).

Apoptosis

Table 1 and Figure 2–5 show AI and stain-ing pattern of apoptosis markers in 19 sectionsof retinoblastoma. Remarkably, the number ofapoptotic figures in almost all cases by far ex-ceeded the number of mitotic figures (Fig. 1B)—the apoptotic paradox.

The mean value of AI was 40.26 (range 1–110, median 39.00). The AI was significantlyrelated to the MI (Mann–Whitney, P = 0.02).Although Bcl-2 expression (Fig. 1D) was in-versely related to AI in several tumors, thedifference between the two groups did notreach significance (Mann–Whitney, P = 0.07).All tumors that showed Bcl-2 overexpres-sion (Bcl-2 positivity ≥ 50%) still showed nu-merous apoptotic cells. In addition, 11 tu-mors (57.89%) did not show Bcl-2 expression(0 percent /Bcl-2- negative staining), and ofthese, four tumors did not show significantapoptosis.

High caspase-3 staining was found in63.15% of the tumors (Fig. 1E), and withinthe total samples, caspase-3 was significantly


Figure 1. Immunohistological features of proliferation and cell death in retinoblastoma.(A). Massive apoptosis in retinoblastoma; apoptotic cells outnumber mitotic figures. (B) MIB-1staining; a high-labeling index as shown here is often seen. (C) Bcl-2 staining; most tumorcells are positive. (D) Fas staining; a coarse membraneous staining is present in this case, butmore often cytoplasmic staining is found. (E1) Staining for activated caspase-3; many positivecells with intact nuclei are seen, suggesting that the number of apoptotic cells is even higherthan appears by morphology; whereas in E2, lower or almost negative caspase-3 staining isseen. Arrows in A show different stages of apoptosis: chromatin condensation (middle arrow),chromatin margination (left arrow), and fragmentation (right arrow). Arrows in E2 show afew of the many apoptotic cells.

related to the AI (Mann–Whitney, P = 0.03).In addition, two tumors with not significant orvery low caspase-3 staining showed a high AI,indicating a caspase-3-independent pathway ofapoptosis.

The AI was not significantly related to p53and Fas/APO-1/CD95 expression (Mann–Whitney P = 0.28 and P = 0.39, respec-tively). P53 positivity was found in 57.89%of tumors; three tumors of p53-negative stain-ing demonstrated a high AI, suggesting a p53-independent apoptotic pathway.

High caspase-3-positive staining was ob-served as a nuclear staining. Figure 5 showsa representative example of a retinoblastomatumor showing caspase-3 immunoreactivity. InFas/APO-1/CD95, the positive staining wasobserved in 12 of 19 tumors. Immunoreactivitywas characterized by a brown predominantlycytoplasmic (not membranous, as expected) re-action product diffusely located throughout thetumors, mainly in the area of rosettes. Only onetumor showed a coarse membranous staining(Fig. 1F).


Figure 2. Staining pattern of apoptotic markersrelated to the AI (n = 19). Box plot of the AI and p53expression (Mann–Whitney U test, P = 0.28).

Figure 3. Staining pattern of apoptotic markersrelated to the AI (n = 19). Box plot of the AI andBcl-2 expression (Mann–Whitney, P = 0.07).

Discussion

Retinoblastoma, although characterized byprogressive growth, consists of a cell popu-lation in which both proliferation and abun-dant cell death occur. Tumor necrosis and celldeath by apoptosis are common features of

Figure 4. Staining pattern of apoptotic markersrelated to the AI (n = 19). Box plot of the AI andcas-3 expression (Mann–Whitney, P = 0.03).

Figure 5. Staining pattern of apoptotic markersrelated to the AI (n = 19). Box plot of the AI and Fasexpression (Mann–Whitney, P = 0.39).

retinoblastoma—in fact so common that, inhistological specimens, apoptotic cells often byfar outnumber mitotic/dividing cells, a pecu-liar phenomenon we call the apoptotic paradox.

The crucial molecular event in retinoblas-toma is loss of both alleles of the RBgene. The RB tumor suppressor restrains


proliferation, in part, by modulating the activ-ity of E2F transcription factors. Consequently,loss of RB tumor suppressor promotes aber-rant proliferation through deregulation of thecell cycle.

Our findings show a marked cell prolifera-tion as evidenced by a high MI and high ex-pression of MIB-1. The high MIB-1 expres-sion in our study (mean 55.26%, range 20–90%) is in keeping with other studies19,20 thathave indicated a high-proliferative capacity ofretinoblastoma.

Although apoptosis is seen in almost all tu-mors, the extent of its occurrence is surpris-ing. In some cases almost half the tumor cellsare undergoing apoptosis. Clearly this wouldpreclude growth of the tumor, and the phe-nomenon is difficult to explain. Perhaps theenucleation procedure triggers massive apop-tosis, although it is strange that other tumorsdo not show this phenomenon upon surgicalremoval. Obviously, this striking feature is fairlytypical of retinoblastoma.

The RB tumor suppressor not only regu-lates proliferation but also influences apoptosisthrough its association with several E2F tran-scription factors. Unrestrained E2F activity, as aconsequence of loss of RB, not only promotes S-phase entry but can also act in concert to induceapoptosis through both p53-dependent and in-dependent mechanisms.21–24 Another gene in-volved in the coupling processes is the c-myc

oncogene.25 When c-myc oncogene is active,there is simultaneous induction of cell prolif-eration and apoptosis. The Myc oncoproteininduces ARF (the alternative reading frameproduct of INK4a/ARF tumor suppressor lo-cus), which in turn activates p53 to trigger celldeath. Consequently, disruption of the ARF-p53 pathway allows inappropriate proliferationand apoptosis, thereby stimulating tumorigen-esis.26–27 The present study showed a link be-tween apoptosis and proliferation, as indicatedby the significant relationship between the MIand AI, although apparently proliferation is fa-vored in dysregulation as there is net growth.This link is in accordance with other studies re-

ported previously.2,28,29 The close relationshipbetween the AI and proliferative index sug-gests that cell proliferation and apoptosis areco-regulated to some degree; and how cell cy-cle progression can be coupled to the apoptoticmachinery.21

p53 Accumulation and Apoptosis

Accumulation of p53 was shown in 14 of 19cases (73.67%). However, our finding of a lackof correlation between the accumulation of p53and AI could be in part explained by the factthat apoptosis is induced through both p53-dependent and independent mechanisms.30–33

Oncogenes can also signal to p53 throughARF-independent mechanisms and promoteapoptosis in ways that are entirely independentof p53. Alternatively, our immunohistochem-ical staining may not reflect the actual statusof the p53 gene. Caution is warranted in in-terpretation, although the cases with very highexpression are likely to have mutated p53.

Bcl-2 Expression and Apoptosis(Caspase-9 or Intrinsic Pathway)

Bcl-2 is widely believed to inhibit apopto-sis, but how Bcl-2 prevents apoptosis remainsunresolved.34,35 It is still unclear whether itsprosurvival members control caspase activationdirectly36 or indirectly by controlling mitochon-drial integrity .

A number of Bcl-2 family members havebeen identified in human cancer. Bcl-2, Bcl-x, Bcl-w, bfl-1, brag-1, mcl-1, and A1 inhibitapoptosis, whereas others, such as Bax, Bik,Bak, Bad, Bid, Bcl-xs, and hrk, activate apopto-sis.37–39 The various Bcl-2 family members candimerize with one another, with one monomerantagonizing or enhancing the function of theother. In this way, the ratio of inhibitors to ac-tivators in a cell may determine if a cell willundergo apoptosis or not.37

Our findings confirmed that in many tumorsoverexpression of Bcl-2 does not always inhibitapoptosis. Several explanations are possible.


First, apoptosis in retinoblastoma is through thecaspase-8 pathway and Bcl-2 expression is anaccidental finding. This so-called death recep-tor pathway is not blocked by overexpressionof Bcl-2 or other anti-apoptotic members ofthe Bcl-2 family.40–42 Second, the Bcl-2 overex-pression does reflect activation of the caspase-9pathway but regulation fails for some reason,for instance, even higher expression of pro-apoptotic family members, such as Bax.

Fas and Apoptosis (Caspase-8or Extrinsic Pathway)

The Fas receptor, a 36-kDa protein andmember of the tumor necrosis factor/nervegrowth factor receptor family, has recently cap-tured the attention of several investigators asan apoptosis-signaling surface receptor that isable to trigger programmed cell death.43,44 Theexpression of Fas has been demonstrated in anumber of solid tumors and hematologic malig-nancies.7 Fas expression did not induce apop-tosis in the majority of tumors in our study.Although Fas expression was seen in 50% ofcases, however, most did not show membranestaining, as expected, but cytoplasmic stain-ing. Only one case showed a clearly positivemembrane staining. Whether this means thatFas is not operative when found only in thecytoplasm or whether this represents an arte-fact of fixation is unclear. Of course, expressionalone is not sufficient for apoptosis. Fas mustbe activated as well, for instance, by Fas ligand.The eye with its “immune-privileged” statusis supposed to be a place were Fas ligand ishighly expressed. So clearly high Fas expres-sion will predispose for apoptosis. Tan et al.45

proposed that Fas receptor activates the down-stream procaspases through a mechanism thatis independent of RB1 cleavage.RB1 cleavagein Fas-induced death may be a bystander eventwith no biological significance, which may ex-plain in part that the Fas receptor-mediatedpathway is not favored in retinoblastomaapoptosis.

Caspase-3 Expression and Apoptosis

RB1 is a target of the caspase family of pro-teases during cell death. C-terminal cleavage ofRB1 by caspase appears to be the first step to-ward, and an important mechanism for, degra-dation and functional inactivation of RB duringcellular apoptosis.46,47 The cleavage and sub-sequent degradation of RB1, in conjunctionwith the elimination of MDM2, leads to theactivation of E2F and p53, which are knownactivators of apoptosis.45 Effector caspase-3 isactivated by the initiator caspase-8 or caspase-9, which produces the characteristic morpho-logical changes associated with apoptosis

Our results show that activation of effec-tor caspase-3 induces apoptosis in humanretinoblastoma, which supports the findingsfound in retinoblastoma nullizygous mice.16 Ina small number of cases, however, no significantstaining with caspase-3 was seen in the pres-ence of massive apoptosis. Although technicalreasons cannot entirely be ruled out, apopto-sis apparently can be induced independentlyon caspase-3 activation, indicating that othercaspase/s may be invoked to induce apopto-sis (caspase-3-independent pathway). This dualapoptotic mechanism, independent and depen-dent on caspase-3, was also observed in centralnervous system neurons proposed by Simpsonet al., 2001.16

Other effector caspases involved in the cas-pase cascade has been reported recently. Inmice, caspase-7, a member of the caspase-3subfamily, was found to cleave (35)S-RB pro-tein at both the carboxyl termini.17 The effec-tor caspase-7 has been proposed to be activatedby initiator caspases48 and may stand in forcaspase-3.

As demonstrated in Table 1 and discussedabove, we did not find any consistent patternin expression of apoptotic and anti-apoptoticmolecules, as indicated by caspase-3, p53, Fas,and Bcl-2 markers. P53 accumulation candownregulate Bcl-2 expression, thereby stimu-lating apoptosis; however, this pattern could befound only in some tumors but not in all cases.


Although this study used only a small numberof markers, the evidence we have found suggestsno preference for a single pathway in apopto-sis in retinoblastoma. Furthermore, from dataabove, it appears that, apart from the two dis-tinct major pathways, a caspase-3-independentpathway seems to be operative in retinoblas-toma, which may represent a cellular attemptto balance uncontrolled tumor proliferation bypromoting apoptosis cell death.

Acknowledgments

The authors wish to thank Dr. P.D. Beze-mer of the Department of Clinical Epidemiol-ogy and Biostatistics, Vrije Universiteit Medi-cal Center for his statistical advice; T. Tademaand W. Vos for technical assistance; EndangRoostini, M.D., Ph.D. of the Department ofPathology, University of Indonesia and Lak-shmi Thaufiq, M.D., University of Padjad-jaran, Indonesia, for providing some speci-mens. R.S. spent a period of research workin the Netherlands supported by the KNAW[the Royal Netherlands Academy of Arts andSciences in the framework of the ScientificProgram Indonesia–Netherlands (SPIN)], theNelly Reef Fund, the Ditmer Funds, and theKWF [KWF-Kankerbestrijding in cooperationwith the Indonesian Cancer Foundation (YKI)and Indonesia Exchange Program (IEP)].

Conflicts of Interest

The authors declare no conflicts of interest.

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