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GASTROENTEROLOGY 2003;125:1094–1104

ow Microvessel Density Is an Unfavorable Histoprognosticactor in Pancreatic Endocrine Tumors

NNE–MARIE MARION–AUDIBERT,* CECILE BAREL,‡ GERALDINE GOUYSSE,*,§

EROME DUMORTIER,*,§ FRANK PILLEUL,� CELINE POURREYRON,* VALERIE HERVIEU,§

ILLES PONCET,‡ CATHERINE LOMBARD–BOHAS,‡ JEAN–ALAIN CHAYVIALLE,*,‡

HRISTIAN PARTENSKY,‡ and JEAN–YVES SCOAZEC*,§

Unite Inserm U45, §Service Central d’Anatomie et Cytologie Pathologiques, ‡Federation des Specialites Digestives, and �Service deadiologie Digestive, Hopital Edouard Herriot, Lyon

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ackground & Aims: In many malignant tumors, intratu-oral microvascular density (MVD) has been suggested

o be a prognostic parameter. We aimed to provide auantitative evaluation of intratumoral microvascularensity in a large series of resected endocrine tumors ofhe pancreas and to evaluate the potential prognosticignificance of this parameter. Methods: Eighty-two tu-ors from 77 patients have been studied. MVD was

valuated by 2 observers after CD34 immunostainingnd correlated with the following parameters: WHO clas-ification, hormonal profile, tumor size, vascular endo-helial growth factor expression, occurrence of metasta-is, duration of survival. Results: MVD ranged from 5 to2 vessels/field. MVD was significantly higher in well-ifferentiated benign endocrine tumors than in tumorsf uncertain behavior and in carcinomas. No close cor-elation was found between MVD and the hormonalrofile. MVD was significantly higher in tumors charac-erized by the following histoprognostic parameters: size

2 cm, proliferation index <2%, no evidence of metas-asis. No close correlation was observed between MVDnd VEGF expression. Finally, a MVD <30 vessels/fieldas associated with the occurrence of metastasis in

umors <2 cm and/or with a proliferation index <2%nd with a significantly shorter survival after surgery.onclusions: The quantitative analysis of microvesselensity in pancreatic endocrine tumors may identifyatients who, despite favorable conventional histoprog-ostic factors, are at risk of unfavorable evolution.

ike normal endocrine tissues, endocrine tumors areusually characterized by a high vascular density, re-

ulting in a hypervascular appearance, which is one ofheir main diagnostic features at imaging studies.1 Theigh vascular density of endocrine tumors is likely toesult from a process of tumor-associated angiogenesis.nimal models have confirmed the existence of this

ngiogenesis process and underlined its role in the de-elopment of endocrine malignancy.2,3 In the well-ex-

lored model of pancreatic endocrine tumorigenesis ob-erved in transgenic mice expressing the SV40 antigennder the control of the insulin promoter,4 the acquisi-ion of angiogenic properties by transformed endocrineells has been shown to occur before the transition fromyperplasia to neoplasia5 and to be a prerequisite for themergence of invasive carcinoma.3 The cellular and mo-ecular mechanisms of tumor angiogenesis in pancreaticndocrine tumors are not fully delineated.6 Normal en-ocrine cells constitutively express potent angiogenicactors, such as VEGF and FGF2.7–10 In animal mod-ls,3,7 as well as in human endocrine tumors,9 no over-xpression of known angiogenic factors has been de-ected, leading to the hypothesis that tumor angiogenesisn pancreatic endocrine tumors may depend on the loss ofxpression of so far unidentified anti-angiogenic or an-iostatic factors present in the normal pancreatic endo-rine tissue.3

Angiogenesis markers have been suggested to be ofrognostic value in tumor pathology.11–13 In a largeumber of malignant tumors, including carcinomas ofreast,14 colon,15 prostate,16 bladder,17 lung,18 and pan-reas,19 the occurrence of a high intratumoral microvas-ular density has been correlated with local invasion,etastatic dissemination, and at least in some series,ith short survival and high risk of recurrence. No

ttempt has been made to evaluate the prognostic valuef angiogenesis markers in human endocrine tumors.his may be of particular relevance, in view of theurrent need for additional histoprognostic factors in thisroup of tumors.

Abbreviations used in this paper: MVD, microvascular density; PBS,hosphate-buffered saline.

© 2003 by the American Gastroenterological Association0016-5085/03/$30.00

doi:10.1053/S0016-5085(03)01198-3

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We therefore aimed to perform a retrospective study ofa large series of resected pancreatic endocrine tumors inorder to: (1) analyze the structural and immunopheno-typic characteristics of intratumoral vessels, and (2) pro-vide a quantitative evaluation of intratumoral microvas-cular density and evaluate the potential prognosticsignificance of this parameter.

Materials and MethodsStudy Group

The study group comprised all patients submitted tosurgical resection for an endocrine tumor of the pancreas atHopital Edouard Herriot between 1986 and 2001 and forwhom both complete clinical information and tissue materialof the primary pancreatic tumor were available. Seventy-sevenpatients fulfilled these criteria. Clinical data were analyzed andthe available imaging data were reviewed. The following in-formation was recorded for each patient: age, sex, location ofthe tumor, hypervascular appearance at the arterial time ofcomputed tomography examination, and/or at arteriography(available in 26 cases), type of treatment, occurrence of metas-tases, duration of follow-up, duration of survival, status at theend of the follow-up period (June 1, 2002) and cause of death.

Tumors were classified according to the WHO criteria20

into the following groups: benign well-differentiated endo-crine tumors, well-differentiated endocrine tumors of uncer-tain behavior, well-differentiated endocrine carcinomas, poorlydifferentiated endocrine carcinomas. The size of the tumor wasthat measured at macroscopical examination of the surgicalspecimen.

The endocrine nature of the tumor was assessed by theimmunodetection of chromogranin A and synaptophysin. Inall cases, the tumor proliferation index was determined bycounting the number of tumor cell nuclei reacting with theMIB-1 antibody, directed against the Ki67 antigen; 1000nuclei have been counted per case; the final result was ex-pressed as the percentage of labeled nuclei in the sample;WHO criteria20 were used to define 4 classes (�2%, 2%–5%,5%–15%, �15%).

The hormonal profile of each tumor was assessed by immu-nohistochemical examination. In all cases, the expression of thefollowing peptides and hormones was tested: insulin, gluca-gon, somatostatin, pancreatic polypeptide, gastrin, calcitonin,serotonin, vasoactive intestinal peptide; the list of antibodiesused for immunohistochemical examination is given in Table1. For each case, were recorded: the hormone(s) detected in ourtechnical conditions and the percentage of tumor cells express-ing each detected hormone. We will hereafter refer to as“non–hormone-producing tumors” all tumors in which, in ourtechnical conditions, none of the hormones tested was detect-able or in which one or several hormones could be detected,but in no more than 5% of tumor cells.

The occurrence of metastases was evaluated as follows:lymph node metastases were assessed by the examination of the

resected surgical specimen, liver metastases were usually diag-nosed at imaging studies and confirmed by either cytologicalor histological preoperative examination in 7 cases or byexamination of a surgical specimen in 13 cases, distant metas-tases were diagnosed at imaging studies. For each patient, thesite and time of occurrence of the metastase(s) were recorded.

Tissue Material

A total of 82 tumors was studied (1 lesion in 73patients, 2 in 3 patients, and 3 in 1 patient). Surgical speci-mens were available in all cases. They were received fresh.When the tumor was visible at external examination, tissuesamples were immediately taken; part of them were frozen inliquid nitrogen and stored; the remainders were fixed in glu-taraldehyde 2.5% for ultrastructural examination; the surgicalspecimen was then allowed to fix. In the other cases, thesurgical specimen was left unopen and was immediately im-mersed in fixative fluid (either Bouin’s fluid or formalin before1997, buffered formalin after 1997). After fixation, sampleswere taken from representative areas of the tumor and at thejunction between tumorous and nontumorous tissue.

Immunohistochemical Study

Antibodies. The list of antibodies used in the study isgiven in Table 1. Several endothelial cell markers21 weretested: (1) von Willebrand factor, an intracellular proteinexpressed by most endothelial cells, (2) CD34, a sialomucintypically expressed by capillary endothelial cells, (3) CD31(PECAM-1), a cell-cell adhesion molecule of the immunoglob-ulin gene superfamily expressed by most endothelial cells, (4)H and Y blood group antigens identified by the monoclonalBNH9 antibody. In addition, we tested the expression ofsmooth muscle isoform of � actin, used as a marker of pericytesand perivascular muscle cells. A polyclonal commercial anti-

Table 1. List of the Antibodies Used in the Study

Clone Source

Chromogranin A polyclonal BoehringerSynaptophysin SVP38 SigmaKi67 MIB-1 DakoInsulin polyclonal ICNGlucagon K79bB10 SigmaSomatostatin polyclonal INSERM U45, LyonPancreatic polypeptide polyclonal INSERM U45, LyonGastrin polyclonal INSERM U45, LyonCalcitonin polyclonal DakoSerotonin 5HT-H209 DakoVIP polyclonal INSERM U45, LyonVon Willebrand factor F8/86 DakoCD34 QBEnd10 DakoCD31 JC/70A DakoH and Y antigens BNH9 Immunotech� smooth muscle actin 1A4 SigmaVEGF polyclonal Santa Cruz

NOTE. Boehringer, Mannheim, Germany; Dako, Glostrup, Denmark;ICN, Costa Mesa, CA; Immunotech, Marseille-Luminy, France; Sigma,St. Louis, MO; Santa Cruz, Santa Cruz, CA.

October 2003 MICROVESSEL DENSITY IN PANCREATIC ENDOCRINE TUMORS 1095

body was used for the detection of VEGF; its specificity hasbeen previously verified.9

Immunoperoxidase technique. An indirect immu-noperoxidase technique was applied to 4 �m-thick deparaf-finized sections. Prior to immunostaining, sections were rehy-drated and incubated in a microwave oven, 3�5 minutes incitrate buffer 10 mmol/L, pH6. After washing in phosphate-buffered saline (PBS), a streptavidin-biotin-peroxidase tech-nique was performed. Briefly, tissue sections were sequentiallyincubated 30 minutes with the primary antibody in appropri-ate dilution, then with biotin-labeled anti-mouse or anti-rabbit immunoglobulin antibody. After incubation with per-oxidase-labeled streptavidin, peroxidase activity was revealedusing diaminobenzidine as a chromogen. For CD34 andMIB-1 immunodetection, all tissue sections were processedduring the same experiment, in an attempt to avoid biases dueto technical artifacts.

Analysis of the results. For evaluation of the appar-ent expression levels of endothelial cell markers, tumor tissuewas compared with normal Langerhans’ islets found in theperitumoral pancreatic parenchyma.

Ultrastructural Study

Glutaraldehyde-fixed tissue samples were processed ac-cording to standard procedures. Briefly, after postfixation inosmium tetroxide, tissue pieces were dehydrated in gradedethanols and propylene oxide, before embedding in epoxyresin. Semi-thin sections were made and stained with toluidineblue. Ultrathin sections were made with a Ultracut E ultra-microtome (Reichert–Jung, Leica Microsystems, Rueil–Mal-maison, France), stained with uranyl acetate and lead citrate,and examined in a Jeol electron microscope (Tokyo, Japan).

Evaluation of Intratumoral MicrovascularDensity

For evaluation of intratumoral microvascular density(MVD), sections of tumor tissue from the primary pancreaticlesion, stained with CD34 antibody, were used. According toprevious recommendations, all independent CD34 positivestructures were counted, irrespectively of the presence of anidentifiable lumen (Figure 1).

To make easier the assessment of inter- and intra-observerreproducibility, numerized images were used. For each tumor,10 consecutive, unselected fields were numerized at a magni-fication of �200 through a Leica photonic microscope (Rueil–Malmaison, France) equipped with a tri-CCD video camera(Nikon, Tokyo, Japan). The corresponding images were storedon a computer; each image was identified by 2 numbers,corresponding respectively to the tumor examined (from 1 to82) and to the field numerized (from 1 to 10 for each tumor).The number of CD34 positive structures in each numerizedimage was then counted independently by 2 observers. Ob-servers were unaware of the correspondence between the num-ber randomly attributed to each tumor and the identity of thecorresponding patient. The mean microvascular density(MVD) for each tumor was calculated as the mean of the counts

performed by the 2 observers, after exclusion of, respectively,the lowest and highest values measured.

To assess intraobserver reproducibility, numerized imagesfrom selected cases were counted 3 times by each observer. Toassess interobserver reproducibility, the 10 counts produced byeach observer for the same tumor were compared.

Evaluation of Microvascular Area andPerimeter

In selected cases, the determination of MVD was as-sociated with the determination of microvascular area andperimeter by a morphometric technique (Scion Software, Fred-erick, MD). 32 tumors were selected for this study: the 10cases with the lowest MVD values, the 10 cases with thehighest MVD values, and 12 randomly selected cases with30�MVD�40 microvessels/field. In the same fields as thosepreviously used for the determination of MVD, we measuredthe surface and perimeter of each CD34� structure. For eachtumor examined, the following measures were recorded: themean area of individual vessels, the total microvascular area/field, corresponding to the sum of the areas of all CD34�structures located in each field, and the mean microvascularperimeter, corresponding to the mean of the perimeters of allCD34� structures identified.

Evaluation of Arteriolar Density

Arterioles were defined as small vessels lined by acontinuous and well formed muscular rim expressing thesmooth muscle isoform of � actin (Figure 1E ). Counts wereperformed according to the same procedure as described pre-viously for evaluation of MVD, in the same numerized images.Intratumoral arteriolar density was expressed as the meannumber of arterioles/field.

Evaluation of VEGF Expression

Two parameters were taken into account for the eval-uation of VEGF expression: the number of tumor cells express-ing the corresponding protein and the apparent level of inten-sity of the immunodetection. As previously described,9 tumorswere classified into three groups according to the number ofVEGF� tumor cells: �25%, 25–50%, and �50%. VEGFexpression was evaluated only in tumor cells. Its apparentintensity level was assessed by comparison with normal Lang-erhans’ islets present in the peritumoral tissue; VEGF expres-sion was considered as weak when the apparent level of ex-pression was fainter in tumor tissue than in normal endocrineislets and strong when expression was comparable in tumortissue and in normal Langerhans’ islets.

Statistical Analysis

Fisher exact test and nonparametric Student t test wereused when appropriate. A P value of less than 0.05 was consideredsignificant. Actuarial survival rates were calculated from the timeof resection until the end of the follow-up period (June 1, 2002),using the Kaplan–Meier method. Only disease-related deaths

1096 MARION–AUDIBERT ET AL. GASTROENTEROLOGY Vol. 125, No. 4

were taken into account for calculation of survival rates. Compar-ison of survival was performed using the log-rank test.

ResultsClinical Characteristics of the Study GroupSeventy-seven patients were included in the study

group. There were 37 male and 40 female. Mean age atresection was 48.3 years (range, 18–88 years). Ninepatients had the MEN1 syndrome. Four patients, includ-ing 3 with MEN1 syndrome, had multiple tumors (2tumors in 3 patients, 3 tumors in 1 patient).

Nineteen patients presented clinical evidence of afunctional hormonal syndrome, related to the hyperse-cretion of insulin in 7 cases, glucagon in 8, vasoactiveintestinal peptide in 1, gastrin in 2, and parathyroid-related hormone in 1. Zollinger–Ellison syndrome waspresent in 1 further patient, with MEN1 syndrome andduodenal gastrinomas.

All patients were submitted to surgical resection: ce-phalic duodenopancreatectomy was performed in 49cases, left pancreatectomy in 21, isthmic resection in 5,and total pancreatectomy in 2. Twenty-one patients re-

Figure 1. Immunohistochemi-cal results. Two examples ofCD34 immunostaining of endo-crine pancreatic tumors areshown in A and B; in A, intratu-moral microvascular density ishigh: CD34� structures are nu-merous and small; in B, intra-tumoral microvascular densityis low: CD34� structures arerare and large. In normal Lang-erhans’ islets (arrow), capillaryvessels are characterized by afaint and heterogeneous ex-pression of CD34 (C) and ofthe smooth muscle isoform of� actin (D). In contrast, thesmooth muscle isoform of � ac-tin is strongly detectable alongintratumoral capillary vesselsand in the wall of arterioles (ar-row) (E). (Immunoperoxidasefollowed by nuclear stainingwith Mayer’s hematoxylin; orig-inal magnifications: A, 200�;B, 200�; C, 350�; D, 350�;E, 200�).


ceived adjuvant chemotherapy (adriamycin-streptozoto-cin, 5FU-streptozotocin, 5FU-deticene, or 5FU-cispla-tin). Five patients received somatostatin analogues and 9received alpha interferon.

Thirty-five patients (45.4%) had evidence of meta-static disease at the time of surgical resection. Peripan-creatic lymph node metastases were present in 14 pa-tients and liver metastases in 21 patients. Distantmetastases were not present at the time of surgical re-section. Ten patients were lost to follow-up. In theremaining 67 cases, the duration of follow up rangedfrom 3 to 228 months (mean � SD � 40.7 � 35).During follow-up, 15 patients presented evidence ofdisease progression. Liver metastases were diagnosed in 6patients with only lymph node metastases at the time ofsurgical resection. Other distant metastases were diag-nosed in 2 patients and peritoneal carcinomatosis in 2. In5 further patients, recurrence of liver metastases after aninitial surgical treatment was documented.

At the end of the follow-up period, 24 patients weredeceased: 15 from their tumor, 8 from postoperativecomplications and 1 from an unrelated cause. Forty-threepatients were alive at the end of the follow-up period; 33patients showed no local recurrence and no evidence ofmetastasis; 10 patients were metastatic. Survival rate was70% at 5 years and 30% at 10 years with a mediansurvival time of 110 months.

Histopathological Characteristics of theStudy Group

Eighty-two tumors were analyzed. 52 tumorswere located in the head of the pancreas, 23 in the tailand 7 in the body. Their size ranged from 5 to 140 mmin diameter (mean � SD � 37.9 � 27.3) (Figure 2).Twenty-one lesions measured less than 20 mm in diam-eter.

According to the WHO criteria,20 23 tumors wereclassified at resection as benign well differentiated endo-crine tumors, 23 as well-differentiated endocrine tumorsof uncertain behavior, 35 as well-differentiated endocrinecarcinomas, and 1 as poorly differentiated carcinoma.The tumor proliferation index, evaluated after immuno-detection of the Ki67 antigen, was: �2% in 54 cases,2%–5% in 13 cases, 5%–15% in 10 cases, and �15% in5 cases. All tumors classified as well-differentiated endo-crine tumors, 18 tumors classified as of uncertain behav-ior, and 13 well-differentiated endocrine carcinomas hada proliferation index �2%. The 5 tumors with a prolif-eration index �15% were 4 cases of well-differentiatedcarcinoma and 1 case of poorly differentiated carcinoma.

By immunohistochemistry, 47 tumors were classifiedas hormone-producing and 34 as non–hormone-produc-ing. 42 tumors expressed at least one of the followingpeptides in more than 10% of tumor cells: glucagon in17 cases, insulin in 9, pancreatic polypeptide in 10,somatostatin in 5, VIP in 1. Five tumors presentedectopic hormone production demonstrated by immuno-histochemistry: gastrin in 2, serotonin in 1, calcitonin in2; an additional case secreted PTH-related hormone,responsible for a clinical functional syndrome and de-tected by serum assay but not demonstrated by immu-nohistochemistry because of the lack of a specific anti-body working in our technical conditions.

Structural and ImmunophenotypicCharacteristics of Intratumoral Vessels

In all cases examined, intratumoral capillary ves-sels present in pancreatic endocrine tumors strongly ex-pressed all the endothelial cell markers tested, includingvon Willebrand factor, CD34 (Figures 1A and 1B),CD31, and BNH9-related antigens. The labeling inten-sity of capillary vessels was higher in the tumor tissuethan in the normal Langerhans’ islets visible in theadjacent pancreatic parenchyma (Figure 1C ). In contrastto the capillary vessels present in the normal Langerhans’islets (Figure 1D), intratumoral capillary vessels wereconstantly lined by cells expressing the smooth muscleisoform of � actin and likely to represent pericytes(Figure 1E ).

Ultrastructural examination, available in 15 cases,confirmed the existence of structural differences betweenintratumoral capillary vessels and the capillary vessels ofnormal Langerhans’ islets. In contrast to the capillaryvessels of the normal endocrine tissue, most intratumoralcapillary vessels were lined by non-fenestrated endothe-lial cells surrounded by a well-formed basement mem-brane. Moreover, in most instances, a perivascular con-

Figure 2. Correlation between intratumoral microvascular density andtumor size.


nective space separated the intratumoral capillary vesselsfrom the adjacent tumor cells.

Intratumoral Arteriolar Density

The mean intratumoral arteriolar density was �1arteriole/field in 34% of tumors, comprised between 1and 2 arterioles/field in 51%, between 2 and 3 arterioles/field in 9%, and �3 arterioles/field in 6%.

Intratumoral Microvascular Density

In our series, intratumoral microvascular density(MVD) ranged from 5 to 92 CD34� structures per field(mean � SD � 35 � 20.4). Taking a 10% differencebetween counts as a threshold value, the concordancebetween 2 readings of the same field was 98% for thesame observer and 96% between the 2 observers.

Correlations Between MVD andMicrovascular Area and Perimeter

In the group of the 10 tumors with the lowestMVD, the mean area of individual vessels was 0.32 �0.15 mm2 and the total vascular area/field was 2.2 � 0.9mm2; the mean perimeter of individual vessels was0.23 � 0.09 mm. In the group of the 10 tumors with thehighest MVD, the corresponding values were respec-tively: 0.11 � 0.05 mm2, 4.3 � 0.8 mm2, 0.14 � 0.06mm. Finally, in the group of 12 tumors with30�MVD�40 microvessels/field, the corresponding val-ues were: 0.19 � 0.09 mm2, 3.5 � 0.8 mm2, 0.17 �0.08 mm.

Comparison Between MVD and ImagingData

Relevant imaging data were available for a retro-spective analysis in 65 patients, for a total number of 68tumors. Tumors were described as hypervascular in 41cases (62%). Their MVD (mean � SD � 45.6 � 16.4)was significantly higher than in tumors without hyper-vascular character at imaging studies (mean � SD �18.4 � 7.8) (P � 0.001). All tumors with MVD�35microvessels/field were described as hypervascular,whereas all tumors with MVD�25 microvessels/fieldwere described as hypovascular, but one. Among the 22tumors with 25�MVD�35 microvessels/field, 12 (55%)were described as hypervascular.

Correlations Between MVD and WHOClassification

MVD (mean � SD) was respectively: 45.7 � 18.4in cases classified as benign well differentiated endocrinetumors, 33.1 � 16.5 in well-differentiated endocrinetumors of uncertain behavior, 25.2 � 17.2 in well-

differentiated endocrine carcinomas; MVD was 6 vessels/field in the only case of poorly differentiated endocrinecarcinoma. MVD was significantly higher in benign tu-mors than in tumors of uncertain behavior and than incarcinomas (Student t test, P � 0.01). Interestingly, alltumors classified as benign well differentiated endocrinetumors had MVD�30 microvessels/field, in contrast to16 of 23 (69.5%) in the group of well-differentiatedendocrine tumors of uncertain behavior and 8 of 35(22.8%) in the group of well-differentiated endocrinecarcinomas. The differences were statistically significant(Fisher exact test, P � 0.01).

Correlations Between MVD and Tumor Size

The corrrelation between MVD and tumor size isshown in Figure 2. The mean MVD was significantlyhigher in tumors measuring less than 2 cm in diameter(Table 2) (Student t test, P � 0.001). MVD was � 30microvessels/field in all tumors less than 20 mm, exceptin 2 cases, both of them metastatic and therefore classi-fied as well differentiated endocrine carcinomas. In con-trast, the 18 tumors measuring more than 50 mm indiameter had a MVD�30 microvessels/field. Among the

Table 2. Correlations Between Mean Microvascular Density(MVD) and Clinical and Histological Data

Numberof cases

MVD(mean � SD) P value

MEN1 syndromePresent 9 40.3 � 15.9 NSAbsent 68 34.5 � 20.6

Functional syndromePresent 19 35.8 � 20.1 NSAbsent 58 32.3 � 17.1

Diameter (mm)�20 mm 21 44.4 � 17.6 P � 0.01�20 mm 61 31.4 � 20.4

Proliferation index�2% 54 41.2 � 19.7 P � 0.001�2% 28 20.1 � 102–5% 13 23.5 � 14.35–15% 10 25.7 � 11.2�15% 5 11.7 � 6.8

Hormonal profileProducing tumors 48 38.3 � 18.6 NSNon-producing tumors 34 30.5 � 22.1

Metastatic disseminationMN 47 43 � 18.4 P � 0.001M� and/or N� 35 24.3 � 17.9M N� 14 22.2 � 9.3M� N�/ 21 25.7 � 9.7

Evolutiona

Alive, M 33 41.2 � 17 P � 0.01Alive, M� 10 20.1 � 6.7

aTen patients lost to follow-up and 9 patients deceased from anunrelated cause were excluded. N, node metastasis; M, distant me-tastasis; NS, not significant.


33 tumors measuring 20 to 50 mm in diameter, 21(64%) had a MVD�30 microvessels/field.

We also compared MVD between tumors measuringmore or less than 30 mm in diameter, since this sizethreshold has been proposed in some studies23–25 todistinguish between benign and malignant tumors.MVD in tumors � 30 mm was significantly higherthan in tumors � 30 mm (Table 3) (Student t test,P � 0.01).

Correlations Between MVD and TumorProliferation Index

The correlations between MVD and tumor pro-liferation index, as evaluated by the immunodetection ofthe Ki67 antigen, are shown in Table 2. The mean MVDwas significantly higher in tumors with a proliferationindex �2% (41.2 � 19.7) than in tumors with a pro-liferation index �2% (20.1 � 10) (P � 0.001). Thelowest value for MVD was observed in tumors with aproliferative index �15% (Table 2).

Correlations Between MVD and HormonalProfile

The mean MVD was slightly higher in patientspresenting with a clinical functional syndrome (mean �SD � 35.8 � 20.1) as compared with those with non-functioning tumors (mean � SD �32.3 � 17.1), but thedifference was not statistically significant (Table 2). Themean MVD was higher in hormone-producing tumors(mean � SD � 38.3 � 18.6), as assessed by immuno-histochemistry, than in nonproducing ones (mean �SD � 30.5 � 22.1) (Table 2). However, the differencewas not statistically significant. We analyzed the patternof vascular density according to the predominant peptidedetected by immunohistochemical techniques. Resultsare indicated in Figure 3. No statistical analysis wasattempted because of the low number of cases included inmost groups.

Correlations Between MVD and Occurrenceof Metastases

The mean MVD was significantly higher in non-metastatic than in metastatic tumors (Student t test, P �0.001) (Table 2). There was no statistically significantdifference between tumors with liver or distant metasta-ses as compared with tumors with only regional lymphnode metastases (Table 2). The mean MVD was signifi-cantly lower in patients with evidence of disease progres-sion during follow-up period (mean � SD � 21.3 � 13),as compared with patients with no evidence of diseaseprogression (mean � SD � 39.4 � 16.1). We verifiedthat, in our series, tumor size was significantly higher(mean � SD � 45 � 24.4 mm) in metastatic than innonmetastatic tumors (mean � SD � 20 � 19.1 mm)(Student t test, P � 0.001). Interestingly, tumor size washigher for tumors with liver metastases (mean � SD �50 � 25 mm) than in tumors with only peripancreaticlymph node metastases (mean � SD � 30 � 22.6 mm).However, the difference did not reach significance levels

Table 3. Correlations Between Mean Microvascular Density (MVD) and Occurrence of Metastasis According to Tumor Size

Diameter (mm) Number of cases MVD (mean) P value Number of cases with MVD � 30 P value

D � 20 mm 21M 19 (90.5%) 46.6 � 17.2 – 0 (0%)M� 2 (9.5%) 24 � 2.8 2 (100%)

D � 20 mm 61M 28 (46%) 39.4 � 19.2 0.003 13 (45%) �0.001M� 33 (54%) 24.3 � 18.5 26 (81.5%)

D � 30 mm 38M 31 (81.5%) 44.3 � 18.5 0.001 8 (26%) �0.001M� 7 (18.5%) 21.3 � 10 6 (86%)

D � 30 mm 44M 16 (36.5%) 37.9 � 18.1 0.05 7 (39%) 0.001M� 28 (63.5%) 25.7 � 19 20 (74%)

Figure 3. Intratumoral microvascular density (MVD) (mean � SD) inpancreatic endocrine tumors according to the hormonal status.


(Student t test, P � 0.11). We then compared MVDbetween metastatic and nonmetastatic tumors within thesame size class. Results are indicated in Table 3. In allthe size classes tested, MVD was significantly higher innonmetastatic than in metastatic tumors.

We verified that, in our series, a high proliferationindex was significantly associated with the occurrence ofmetastases: only 24% of the tumors with Ki67 index�2% were metastatic as compared with 78.5% of tu-mors with Ki67 index �2% (P � 0.01). We thencompared MVD between metastatic and nonmetastatictumors within the same tumor proliferation index class.Results are indicated in Table 4. In all the classes tested,MVD was higher in nonmetastatic than in metastatictumors. When the number of cases included in each classwas sufficient to allow statistical analysis, the differencewas statistically significant.

Correlations Between MVD and Evolution

Tumor MVD was significantly higher in patientsalive without metastasis at the end of the follow-upperiod than in patients alive with metastatis (Table 2)(Student t test, P � 0.01). There was no statisticallysignificant difference in tumor MVD between patientsalive or deceased at the end of the period of follow-up.However, patients with tumor MVD�30 microvessels/field had significantly longer survivals than patients withtumor MVD�30 (log-rank test, P � 0.03) (Figure 4).We verified that, in our series, the presence of metastasiswas associated with a significantly shorter survival (log-rank test, P � 0.03). However, we did not detect anystatistically significant difference in survival according totumor size.

VEGF Expression

Four patterns of VEGF expression were observed:(1) expression in rare, scattered tumor cells (27% of

cases), (2) weak expression in �50% of tumor cells(39%), (3) strong expression in 25%–50% of tumor cells(19.5 %), (4) strong expression in �50% of tumor cells(14.5%). A strong VEGF expression was observed in61% of cases classified as benign well-differentiated en-docrine tumors, 48% of cases classified as well-differen-

Table 4. Correlations Between Mean Microvascular Density (MVD) and Occurrence of Metastasis According to the TumorProliferation Index

Proliferative index (%) Number of cases MVD (mean) P value Number of cases with MVD � 30 P value

�2% 54M 41 (76%) 47.2 � 17.7 �0.001 11 (27%) �0.001M� 13 (24%) 21.9 � 11.6 7 (54%)

�2% 28M 6 (21.5%) 32.5 � 15.4 0.02 3 (50%) �0.001M� 22 (78.5%) 18.9 � 8.3 20 (91%)

�5% 15M 3 (20%) 30.6 � 12.2 NS 1 (33%) NDM� 12 (80%) 17.8 � 10 10 (83%)

�15% 5M 0 (0%) – ND – NDM� 5 (100%) 11.7 � 6.9 5 (100%)

NS, not significant; ND, not done.

Figure 4. Survival curves (according to Kaplan–Meier) for all thepatients of the study group according to intratumoral microvasculardensity � 30 (open circles) or � 30 (open squares); P � 0.05(log-rank test).


tiated tumors of uncertain behavior, and 8% of casesclassified as endocrine carcinomas.

According to the pattern of VEGF expression, intratu-moral MVD (mean � SD) was: 16.1 � 8 microvessels/field(range, 5–32) in cases with only scattered VEGF-positivecells, 26 � 17.9 microvessels/field (range, 10–74) in caseswith weak VEGF expression in �50% of tumor cells,38.7 � 16 microvessels/field (range, 18–70) in cases withstrong VEGF expression in 25%–50% of tumor cells, 71 �14.9 microvessels/field (range, 62–92), in cases with strongVEGF expression in �50% of tumor cells.

DiscussionOur results show that, in endocrine tumors of the

pancreas, intratumoral microvascular density is highlyvariable and suggest that this parameter may be ofprognostic value: in our series, patients with tumors oflow microvascular density are at higher risk of metastasisand present shorter survivals after surgical resection thanpatients presenting with tumors of high microvasculardensity.

In our experience, the intratumoral microvascular den-sity of endocrine pancreatic tumors shows a very largerange of variation, from 5 to 92 microvessels (identifiedas CD34� structures) per field. This finding is somewhatunexpected. Like other endocrine tumors and in contrastto pancreatic adenocarcinomas, pancreatic endocrine tu-mors are usually hypervascular at imaging studies.26–28 Itmight have therefore been expected that MVD values inpancreatic endocrine tumors would be constantly high.In contrast, a significant proportion of the tumors exam-ined in our study presented with MVD values compara-ble to, or even lower than, those usually measured inpancreatic adenocarcinomas.19,29,30 In 41 cases (50%),MVD was �30 vessels per field and in 9 cases (11%),MVD was even less than 15 vessels per field. The retro-spective review of available imaging data suggests a goodcorrelation between the microvascular density evaluatedby histological techniques and the actual tumor perfu-sion evaluated by imaging studies. However, this pointneeds to be further explored by prospective studies usingstandardized imaging techniques.

Variations in intratumoral microvascular density areassociated with differences in the overall organization ofthe vascular supply. Our morphometric analysis revealsthat, in tumors with low MVD, vascular sections wereusually large, as shown by the comparatively high figuresobtained for, respectively, microvascular area and micro-vascular parameter. In contrast, tumors with high MVDcontain numerous small microvessels. In consequence, inour series, the variations in the vascular area, i.e., the

percentage of the tumor tissue occupied by vascularsections, were less marked than the variations in mi-crovessel density. However, the 2 parameters remaincorrelated. We verified that, in our series, the arteriolarsupply of endocrine pancreatic tumors, in contrast totheir microvascular supply, presented no significant vari-ation according to microvessel density, tumor size, orimaging characteristics.

In addition to quantitative variations, our findingsalso point to the existence of qualitative alterations in thestructural and immunophenotypic characters of the vas-cular supply of pancreatic endocrine tumors as comparedwith the normal vascular supply of Langerhans’ islets. Incontrast to normal capillary vessels, intratumoral capil-lary vessels were characterized by a strong and constantexpression of the continuous endothelial cell markers andby a strong immunoreactivity for the smooth muscleisoform of � actin, suggesting the presence of numerouscontractile perivascular cells. Ultrastructural examina-tion, available in 15 cases, showed that most intratu-moral capillary vessels lacked the intra-cytoplasmic fen-estrations characteristic of the capillary vessels associatedwith normal Langerhans’ islets.31–34 Our observationstherefore suggest that, in endocrine tumors, tumor-asso-ciated capillary vessels lack the differentiated featurescharacteristic of the capillary vessels present in normalendocrine tissues. Comparable observations have beenperformed in other types of malignant tumors arising intissues containing highly specialized capillary vessels,such as the liver35 and the brain.36

In most studies, a high intratumoral microvasculardensity has usually been correlated with adverse his-toprognostic features, occurrence of local invasion andincreased risk of metastatic dissemination.11–13 Unex-pectedly, our results suggest that, in endocrine tumors ofthe pancreas, the reverse is true. Indeed, in our series, ahigh MVD was strongly associated with factors known tobe associated with a good prognosis, including small size,low tumor proliferation index, and absence of metasta-sis.23–25,37–40 One hypothesis is that small, well-differen-tiated endocrine tumors of the pancreas are able to inducethe maintenance of organoid features in their microen-vironment, including a high microvessel density recall-ing the situation in the normal pancreatic endocrinetissue.

In order to test the potential prognostic value ofMVD, we analyzed its correlations with the histoprog-nostic factors retained in the recent WHO classification,i.e., size, proliferation index, and evidence of local inva-sion and/or metastatic dissemination.20 We verified that,in our series, intratumoral MVD was significantly higher


in tumors of small size (�2 cm or �3cm according tothe threshold considered) as compared with larger ones,and in tumors with low proliferation index (�2% ac-cording to the threshold retained by the recent WHOclassification20). We also verified that intratumoral MVDwas significantly higher in nonmetastatic tumors than inmetastatic ones. Interestingly, we were also able to showthat, in each size or proliferation index class considered,MVD was significantly lower in metastatic tumors thanin nonmetastatic ones. This strongly suggests that MVDmay be an additional prognostic factor useful to identifypatients at risk for metastatic dissemination, even withtumors of small size and/or low proliferation index.

Some of our results suggest that a threshold of 30 mi-crovessels/field may be retained for prognostic purposes.MVD was �30 microvessels/field in 100% of the tumorsclassified as benign according to the WHO criteria, ascompared with 69.5% of the cases classified as well differ-entiated endocrine tumors of uncertain behavior and only22.8% of the cases classified as endocrine carcinomas. Ineach size or proliferation index class, the number of tumorswith MVD�30 microvessels/field was significantly higherin the metastatic group than in the nonmetastatic one.Finally, we could demonstrate that, in our series, patientshaving tumors with MVD�30 microvessels/field had sig-nificantly shorter survival. Our findings therefore stronglysuggest that MVD may prove to be useful for identifyingpatients with poor prognosis. However, our current results,drawn from a retrospective study including only selectedpatients submitted to surgical resection, must be verified infurther prospective studies, based on larger series. Suchstudies are also needed to determine whether the prognosticvalue of MVD may vary according to the site of origin ofendocrine tumors. An inverse correlation between size andvascular density, as observed here in pancreatic endocrinetumors, has been previously reported in prolactinomas.41 Incontrast, recent studies suggest that, in rectal carcinoids,42 ahigh MVD is associated with poor prognosis. This sug-gests that the cellular and molecular mechanisms oftumor-associated angiogenesis may vary according to thesite of origin of endocrine tumors. This is in line withprevious experimental results43 underlining the impor-tance of host microenvironmental factors in the regula-tion of endocrine tumor cell growth and differentiation.

Previous studies9,44 have shown that VEGF, a potentangiogenic factor constitutively expressed by many nor-mal endocrine cells, is frequently underexpressed or evenundetectable in human enteropancreatic endocrine tu-mors. In line with these previous observations, VEGFexpression was weakly or barely detectable in 66% of ourcases. Moreover, in accordance with recent results,44 we

found no close correlation between VEGF expressionand MVD. Other factors may therefore be involved inthe regulation of tumor-associated angiogenesis. Sinceseveral hormonal peptides may have either pro-angio-genic45,46 or anti-angiogenic47,48 properties, we at-tempted to analyze the variations in the microvasculaturepattern of pancreatic endocrine tumors according to theirhormonal profile. In our series, MVD was higher infunctioning and/or hormone-producing tumors, but thedifferences did not reach significance levels. Large vari-ations in MVD were observed according to the nature ofthe predominant hormone secreted; however, numbers ineach category were too low to allow any relevant statis-tical analysis. In conclusion, our study has revealed anunexpected degree of heterogeneity in the vascular sup-ply of the endocrine tumors of the pancreas and stronglysuggests that its quantitative analysis may provide addi-tional information of prognostic value.

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Address reprint requests to Jean-Yves Scoazec, M.D., Anato-mie Pathologique, Hopital Edouard Herriot, 69437 Lyon, Cedex03, France. e-mail: [email protected]; fax: (33)472116891.

Supported by grants from Ligue contre le Cancer (Comite de Saoneet Loire), Region Rhone-Alpes (Programme Emergence), Societe Na-tionale Francaise de Gastro-Enterologie (Fonds de Recherche), andFondation pour la Recherche Medicale. Anne-Marie Marion-Audibert isa recipient of a grant from Fondation de France. Geraldine Gouyssewas supported by a grant from Hospices Civils de Lyon (Appel d’OffresRecherche Clinique 1997).


low microvessel density is an unfavorable histoprognostic factor in pancreatic endocrine tumors

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