High-grade glioma before and after treatment with radiation and Avastin: Initial observations

700Neuro-Oncology■JULY 2006

Neuro-oNcology

We evaluate the effects of adjuvant treatment with the angiogenesis inhibitor Avastin (bevacizumab) on path-ological tissue specimens of high-grade glioma. Tissue from five patients before and after treatment with Avastin was subjected to histological evaluation and compared to four control cases of glioma before and after similar treatment protocols not including bevacizumab. Clinical and radiographic data were reviewed. Histological analy-sis focused on microvessel density and vascular mor-phology, and expression patterns of vascular endothe-lial growth factor–A (VEGF-A) and the hematopoietic stem cell, mesenchymal, and cell motility markers CD34, smooth muscle actin, D2-40, and fascin. All patients with a decrease in microvessel density had a radiographic response, whereas no response was seen in the patients with increased microvessel density. Vascular morphol-ogy showed apparent “normalization” after Avastin treatment in two cases, with thin-walled and evenly dis-tributed vessels. VEGF-A expression in tumor cells was increased in two cases and decreased in three and did not correlate with treatment response. There was a trend toward a relative increase of CD34, smooth muscle actin, D2-40, and fascin immunostaining following treat-

High-grade glioma before and after treatment with radiation and Avastin: Initial observations

Ingeborg Fischer, Clare H. Cunliffe, Robert J. Bollo, Shahzad Raza, David Monoky, Luis Chiriboga, Erik C. Parker, John G. Golfinos, Patrick J. Kelly, Edmond A. Knopp, Michael L. Gruber, David Zagzag, and Ashwatha NarayanaDepartments of Pathology (I.F., C.H.C., L.C., D.Z.), Neurosurgery (R.J.B., E.C.P., J.G.G., P.J.K.), Radiology (D.M., E.A.K.), Oncology (M.L.G.), and Radiation Oncology (S.R., A.N.), New York University Medical Center, New York, NY, USA

Received January 8, 2008; accepted March 17, 2008.

C.H.C. and R.J.B. contributed equally to this article.

Address correspondence to Ingeborg Fischer, The University of Texas MD Anderson Cancer Center, Department of Pathology, 1515 Holcombe Blvd., Houston, TX 77030, USA (ifischer@mdanderson .org).

ment with Avastin. Specimens from four patients with recurrent malignant gliomas before and after adjuvant treatment (not including bevacizumab) had features dis-similar from our study cases. We conclude that a change in vascular morphology can be observed following anti-angiogenic treatment. There seems to be no correlation between VEGF-A expression and clinical parameters. While the phenomena we describe may not be specific to Avastin, they demonstrate the potential of tissue-based analysis for the discovery of clinically relevant treatment response biomarkers. Neuro-Oncology 10, 700–708, 2008 (Posted to Neuro-Oncology [serial online], Doc. D08-00006, August 12, 2008. URL http://neuro-oncology .dukejournals.org; DOI: 10.1215/15228517–2008–042)

Keywords: angiogenesis, Avastin, bevacizumab, bio-markers, glioblastoma, glioma

Angiogenesis mediated by vascular endothelial growth factor (VEGF) is instrumental to the growth and progression of high-grade gliomas.1

Avastin (bevacizumab) is a monoclonal antibody that binds to VEGF-A, blocking its receptor interactions. In preclinical trials, Avastin has been shown to inhibit angiogenesis, decrease microvascular density and vascu-lar permeability, and, most importantly, inhibit tumor growth.2–5 No tissue-based data on the effect of anti-angiogenic treatment in patients with glioma have been reported.

Avastin is currently being tested in phase II clinical trials for treatment of recurrent malignant gliomas. We

Copyright 2008 by the Society for Neuro-Oncology

Fischer et al.: Glioma before and after Avastin

Neuro-oNcology • o C T o B E R 2 0 0 8 701

have observed a heterogeneous clinical and radiographic response in a cohort of recurrent high-grade gliomas treated with Avastin at our institution. Of interest, some patients experienced multifocal tumor recurrence fol-lowing treatment with Avastin.

Such variable clinical outcomes underscore the impor-tance of understanding correlations between pathologi-cal and clinical features and treatment response. To this end, analysis of tumor tissue is an important tool to vali-date treatment targets and identify potential biomark-ers of treatment response. In this study, we describe the histological features of five cases of recurrent high-grade glioma before and after Avastin treatment.

Materials and Methods

Patients who underwent stereotactic needle biopsy or craniotomy and resection by the Department of Neuro-surgery at New York University (NYU) Medical Center following Avastin treatment were retrospectively identi-fied from a collective of patients with recurrent high-grade gliomas treated with Avastin as a salvage therapy at NYU Medical Center.

A retrospective chart and imaging review was con-ducted including all inpatient hospital charts and out-patient clinical data from the treating neurosurgeons, neuro-oncologists, and radiation oncologist.

Images were retrieved from the electronic archive of the NYU Department of Radiology. A retrospective analysis of the radiological imaging was performed, focusing on the immediate pretreatment and subsequent posttreatment MR imaging. Imaging was performed on a 1.5T system (Avanto, Sonatavision, or Symphony; Sie-mens, Erlangen, Germany). Conventional MR imaging, including nonenhanced axial T1-weighted spin-echo, axial T2-weighted, axial fluid-attenuated inversion recovery, and postcontrast axial T1-weighted imag-ing, was analyzed. Gradient-echo planar images were acquired during the first pass bolus of gadopentetate dimeglumine (Magnevist; Berlex Laboratories, Wayne, NJ, USA), injected at a rate of 5 ml/sec. A total of 60 images were acquired at 1-second intervals. Regions of interest (ROIs) were obtained in both abnormally enhancing tissues as well as abnormal, nonenhanc-ing areas of T2 and FLAIR hyperintensity. Paraffin- embedded tumor specimens were obtained from the archives of the Department of Pathology. Sections stained with hematoxylin and eosin (H&E) were retro-spectively reviewed for selection of blocks for immuno-histochemistry. All immunostains were performed on formalin-fixed, paraffin-embedded tumor tissue with a Ventana automated immunostainer. Primary antibod-ies included VEGF-A (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD34 (Ventana, Tucson, AZ, USA), smooth muscle actin (Ventana), fascin (Ventana), colla-gen IV (Ventana), and D2-40, also known as podoplanin (Signet Laboratories, Dedham, MA, USA).

For comparison of histological features and immuno-histochemistry, four control cases of high-grade gliomas before and after adjuvant treatment without Avastin

were randomly selected from the archives of the Depart-ment of Pathology, Division of Neuropathology at NYU Medical Center.

Histological evaluation was performed by two inde-pendent observers (I.F., C.H.C.) using a standard light microscope. Microvascular density was determined by CD34 immunostains as described previously.6 Briefly, the areas of highest microvascular density on CD34-stained sections were first identified at scanning power. Microvessel counts were subsequently performed in the selected areas with a 203 objective (total magnification 2003). Each stained lumen as well as each CD34-stained single endothelial cell was counted as a microvessel. The results were expressed as the highest number of micro-vessels within any single 2003 microscopic field.

VEGF, CD34, smooth muscle actin, D2-40, and fascin expression in tumor cells was evaluated by esti-mating the percentage of cells labeled by each stain in the whole tissue section as well as cells labeled in the “hot spot” 403 high-power field in which most cells are labeled as determined at scanning power (1.253). The percentages were averaged and assigned semiquantita-tive scores according to the percentage of cells labeled as follows: “0” (no labeling of tumor cells), “11” (less than 1%), “21” (1%–10%), “31” (.10%–50%), and “41” (.50%). The scores were used for comparison of pretherapy with posttherapy expression levels of each immunohistochemical marker (Table 1).

Results

Clinical

The clinical data are summarized in Table 2. Five patients aged 29 to 59 years (mean, 47 years) at the time of initial diagnosis underwent resection or biopsy of a high-grade glioma before and after treatment with Avastin. Four of these patients had recurrent tumors and underwent prior resections. Histological diagnoses were anaplastic oligoastrocytoma (one patient), glioblastoma (two), anaplastic oligodendroglioma (one), and anaplas-tic astrocytoma (one). Four patients had a high-grade glioma (WHO grade III or IV) at initial presentation, and one patient experienced secondary progression of a low-grade glioma 5 years after initial pathological diag-nosis. The time interval from initial diagnosis to start of Avastin treatment ranged from 1 month to 71 months.

All patients received a total dose of 59.4 Gy of frac-tionated, external-beam involved-field radiotherapy. Four patients treated with Avastin for recurrent high-grade glioma underwent radiation as part of their initial therapy. All patients previously received chemotherapy, consisting of four cycles of temozolomide (Temodar) and carboplatin either as single-agent treatment or in com-bination (Table 2). One patient received Avastin as part of the initial chemotherapy protocol concomitantly with radiation.

Karnofsky performance scores were 90 or greater in all patients when Avastin was started. Patients 1–4 had a partial response to treatment by Macdonald cri-


702 Neuro-oNcology • o C T o B E R 2 0 0 8

matter, genu of the corpus callosum, and left basal gan-glia (Fig. 1c).

In patient 2, the pretreatment scan demonstrated prior bifrontal craniotomy with nodular enhancing tumor at the surgical cavity margins and T2/FLAIR signal abnormalities in the frontal lobes and bilateral anterior temp lobes (Fig. 2a). Posttreatment scan dem-onstrated an initial interval response to therapy, with a decreased size of the nodular enhancing component. There was also an interval decrease in the perilesional T2/FLAIR hyperintense signal in the frontal lobes (Fig. 2b). Four months later, there was a marked increase in left frontal and temporal lobe enhancement, with inter-val extension of abnormal T2/FLAIR hyperintense sig-nal and associated mass effect to the left basal ganglia and caudate (Fig. 2c).

In both patients 3 and 4, pretreatment images revealed postoperative resection cavities with a rim of contrast enhancement and surrounding T2/FLAIR sig-nal abnormality, which initially decreased following Avastin treatment. Subsequent interval imaging over the following weeks and months demonstrated progressive enlargement of both the enhancing and nonenhancing portions of the tumor at the margins of the resection cavity consistent with local tumor progression.

Patient 5 had two adjacent peripherally enhancing masses in the right frontal lobe; abnormal T2/FLAIR signal was evident involving the entire right frontal lobe white matter and extending across the midline via the genu of the corpus callosum. Following resection, radiotherapy, and chemotherapy, including treatment with Avastin, there was increased nodular enhancement within the genu of the corpus callosum, internal capsule, insular cortex, with a larger heterogeneously enhancing, and a new mass within the right paramedian frontal lobe, consistent with multifocal recurrence.

teria,7 whereas patient 5 had progressive disease. At the time of recurrence after treatment with Avastin, tumors involved more than one lobe of the cerebral hemispheres in all patients. Three patients had local and two patients (patients 1 and 5) had multifocal disease progression on MRI images (Figs. 1 and 2). The interval between the initiation of Avastin therapy and the post-Avastin tis-sue biopsy or resection was 18–61 weeks, and the inter-val between last Avastin dose and post-Avastin tissue biopsy or resection ranged from 16 to 208 days (Table 2). One patient underwent a stereotactic biopsy, two patients a partial, one patient a subtotal, and one patient a gross total resection of the contrast-enhancing tumor portions.

In the control group, patient age ranged from 42 to 62 years. There were two males and two females. His-tological diagnoses before and after adjuvant radiation and chemotherapy were glioblastoma (two), anaplastic oligodendroglioma (one), and anaplastic oligoastrocy-toma (one).

Imaging

In patient 1, pretreatment scans (Fig. 1a) demonstrated prior left frontoparietal craniotomy and peripheral contrast enhancement around a resection cavity. There was no nodular or mass-like enhancement. After treat-ment (Fig. 1b), there was persistent minimal peripheral enhancement and increased periventricular and left fron-toparietal hyperintense T2/FLAIR signal. Two months later, there was multifocal progression with multiple new enhancing nodules and a dominant right frontal lobe heterogeneously enhancing mass with a marked increase in surrounding T2/FLAIR hyperintense signal consistent with a combination of vasogenic edema and/or infiltrative tumor, extending into the frontal white

Table 1. Summary of comparative immunohistochemistry results in study and control group patients. Recorded is the comparison of the post-Avastin to the pre-Avastin findings for each histological parameter for each patient. Increases are highlighted in light gray, decreases in dark gray. An increase or decrease in staining is defined as a change in the semiquantitative score.

Histology Immunohistochemistry MVD CD34 VEGF-A D2-40 Fascin SMA

Patient 1 Decrease Increase Decrease Increase Increase Increase

Patient 2 Decrease Increase Increase Increase Same Same

Patient 3 Decrease Same Increase Same Same Same

Patient 4 Decrease Same Decrease Same Decrease Same

Patient 5 Increase Same Decrease Same Same Same

Control 1 Decrease Same Increase Decrease Increase Same

Control 2 Decrease Decrease Increase Increase Increase Same

Control 3 Decrease Same Decrease Decrease N/A N/A

Control 4 Increase Increase Decrease Decrease Decrease Increase

Abbreviations: MVD, microvessel density; VEGF-A, vascular endothelial growth factor–A; SMA, smooth muscle actin; N/A, not available.



Tab

le 2

. Clin

ical

dat

a

La

st

Init

ial

Ava

stin

N

umbe

r

His

tolo

gica

l A

djuv

ant

KPS

D

iagn

osis

to

Ex

tent

of

Adj

uvan

t

of

Pa

tien

t A

ge/S

ex

Loca

tion

D

iagn

osis

Tr

eatm

ent

Ava

stin

to

Ava

stin

a Su

rger

y R

esec

tion

b C

hem

othe

rapy

R

espo

nsec

Prog

ress

iond

Res

ecti

ons

1 29

/F

Left

fro

ntal

A

napl

astic

PB

RT

90

2 m

os.

77 d

ays

Biop

sy

Tem

ozol

omid

e 1

Yes

Mul

tifoc

al

3

olig

oast

rocy

tom

a

A

vast

in (

4 cy

cles

)

2 55

/F

Left

fro

ntal

G

liobl

asto

ma

59.4

Gy

1

100

26 m

os.

16 d

ays

Part

ial

Irin

otec

an 1

Ava

stin

Ye

s M

ultif

ocal

4

carb

opla

tin

(5 c

ycle

s), A

vast

in

(4 c

ycle

s)

alon

e (3

cyc

les)

3 59

/M

Left

fro

ntal

A

napl

astic

59

.4 G

y 1

9

0 6

mos

. 93

day

s G

ross

tot

al

Irin

otec

an 1

Ava

stin

Ye

s Lo

cal

2

astr

ocyt

oma

Tem

odar

(6

cyc

les)

, car

bopl

atin

(4

cyc

les)

1

Ava

stin

(4

cycl

es)

4 45

/M

Rig

ht f

ront

al

Ana

plas

tic

59.4

Gy

1

100

43 m

os.

148

days

Su

btot

al

Irin

otec

an 1

Ava

stin

Ye

s Lo

cal

4

olig

oden

drog

liom

a ca

rbop

latin

(2

cyc

les)

(4

cyc

les)

5 47

/M

Rig

ht f

ront

al

Glio

blas

tom

a 59

.4 G

y 10

0 1

mo.

20

8 da

ys

Part

ial

Tem

odar

1 A

vast

in

No

Mul

tifoc

al

2

(4 c

ycle

s)

Abb

revi

atio

ns: F

, fem

ale;

PBR

T, p

roto

n be

am r

adia

tion

the

rapy

; mo.

, mon

th; M

, mal

e.

a Dia

gnos

is t

o A

vast

in r

efer

s to

dia

gnos

is w

ith

high

-gra

de g

liom

a.

b Par

tial

res

ecti

on: ,

50%

con

tras

t-en

hanc

ing

tum

or v

olum

e, s

ubto

tal 5

0%–9

9%, g

ross

tot

al 1

00%

.

c Den

otes

a p

arti

al r

adio

grap

hic

resp

onse

to

Ava

stin

the

rapy

.

d Loc

al p

rogr

essi

on: n

ew c

ontr

ast

enha

ncem

ent

at t

he m

argi

n of

the

res

ecti

on c

avit

y; m

ulti

foca

l pro

gres

sion

: mul

tilo

bar,

bila

tera

l enh

ance

men

t or

mul

tilo

bar

T2

hype

rint

ense

sig

nal.



Histology

Immunohistochemical findings are summarized in Table 1. Cases 1 and 2 are depicted in Figures 3 and 4. In all cases, morphological tumor appearance was similar before and after treatment. The histological diagnoses were identical, except for one patient whose tumor had been diagnosed as anaplastic astrocytoma but had pro-gressed to glioblastoma following adjuvant treatment. Radiation effect consisting of thickened and hyalinized vessel walls was present in all patients. A sarcomatous tumor component consisting of fascicles of spindle cells separating nodules of round tumor cells with moderate amounts of surrounding cytoplasm could be observed in both resection specimens from one patient. There was no morphologically sarcomatous differentiation in the remaining five patients or in the control group.

Vascular Morphology and VEGF Expression

In all cases, blood vessels displayed typical features of highly angiogenic tumors: all had rounded endothelial cells with enlarged nuclei and clustering of blood ves-sels in groups, often decorated by vascular arcades and/or glomeruloid proliferations. Following treatment with Avastin, the appearance of the vasculature was similarly bizarre in three study and all control group cases. How-ever, the glioblastomas in patients 1 and 2 were outfitted with a network of regularly spaced, thin-walled, some-times dilated blood vessels that no longer included vas-cular clusters, arcades, or glomeruloid structures (Fig. 3f). The microvascular density was decreased on the sec-ond resection in all cases, except for the tumor of patient 5, where the microvessel count was increased.

A variable proportion of tumor cells displayed cyto-plasmic staining with VEGF-A. Comparison of VEGF-A expression in tumors before and after Avastin therapy

Fig. 1. T2- (left) and T1- (right) weighted MRI images of patient 1. (a) Resection cavity with peripheral contrast enhancement. (b) Per-sistent minimal peripheral enhancement and increase of periven-tricular and left frontoparietal T2 signal. (c) Multiple enhancing nodules and a dominant right frontal lobe mass with markedly increased surrounding T2 hyperintense signal.

Fig. 2. T2- (left) and T1- (right) weighted MRI images of patient 2. (a) Nodular enhancing tumor around surgical cavity margins and T2 signal abnormalities in the frontal lobes and bilateral anterior tem-poral lobes. (b) Decrease of the nodular enhancing component and frontal T2 signal intensity. (c) Increase in left frontal and temporal lobe enhancement and new involvement of the left basal ganglia and caudate with increased T2 signal and increased mass effect.



demonstrated a relative increase in expression in three cases and a decrease in two cases. Increased VEGF-A expression levels were seen in two cases after treatment without Avastin, whereas the other two cases showed decreased VEGF-A expression. Vascular basement mem-branes were also outlined by VEGF-A immunostains in all cases (Figs. 3b, 3g, 4b, and 4g).

Collagen IV immunostains were available for analysis in two cases, decorating a continuous ring of basement membrane as an integral part of vessel walls both before and after adjuvant treatment (not shown).

Mesenchymal and Hematopoietic Stem Cell and Invasion Markers

A CD34 immunostain labeled an increased proportion of tumor cells, often surrounding blood vessels, following Avastin treatment in patients 1 and 2 (Figs. 3f and 4f). The tumors of the remaining three patients did not have

any labeling of tumor cells. Three of four control cases also included tumor cells reactive with CD34. However, the relative proportion of labeled cells was decreased in two cases and unchanged in the third.

Smooth muscle actin immunostains marked pericytes surrounding blood vessels in all tumors (Figs. 3e and 4e), and an estimate of the percentage of CD34-labeled blood vessels also highlighted by smooth muscle actin did not reveal any difference between consecutive resec-tion specimens. There was also cytoplasmic and mem-branous staining of tumor cells with smooth muscle actin in five cases (Fig. 3j), including two cases of the control group. The percentage of tumor cells stained was increased in patient 1 and in one patient in the control group.

D2-40 (podoplanin) revealed staining patterns simi-lar to smooth muscle actin, decorating the cytoplasm and cell membranes of some tumor cells, with staining accentuated around tumor blood vessels. In contrast

Fig. 3. Histological findings in patient 2 (upper row before treatment, lower row after treatment). (a, f) CD34 highlights vascular arcades prior to Avastin and increased tumor cell staining after. (b, g) Vascular endothelial growth factor–A in vascular basement membranes and rare tumor cells. (c, h) D2-40 expression is more diffuse after treatment. (d, i) Fascin highlights a similar percentage of tumor cells before and after treatment. (e, j) Smooth muscle actin labels vascular smooth muscle cells before and the majority of tumor cells after treatment. original magnification: 403.

Fig. 4. Histological findings in patient 1 (upper row before treatment, lower row after treatment). (a, f) CD34 immunostain highlights glom-eruloid proliferations; after treatment, tumor cells are also labeled. (b, g) Vascular endothelial growth factor–A immunostain labels basement membranes of tumor blood vessels. (c, h) D2-40 expression is increased after Avastin treatment. (d, i) Fascin highlights invading tumor cells. (e, j) Smooth muscle actin labels only vascular smooth muscle cells before but tumor cells after treatment. original magnification: 403.



to smooth muscle actin, all tumors including the con-trol group displayed immunoreactivity for this marker. The proportion of tumor cells labeled was similar or increased following adjuvant therapy in the Avastin group (Figs. 3h and 4h) but decreased in three of four cases in the control group.

Fascin labeled a variable percentage of tumor cells prominently at the invading edge of the tumor, where available for evaluation (Figs. 3c, 3h, 4c, and 4h), but no particular trend in expression levels after treatment was found.

Discussion

Inhibitors of angiogenesis are a promising tool for the treatment of high-grade gliomas. The clinical response to these agents is variable, and presently there are no criteria for optimal patient selection. Furthermore, no data exist on changes that occur in tumors following anti angiogenic treatment. This is perhaps because a neurosurgical procedure with associated morbidity is rarely indicated following salvage therapy for recurrent high-grade gliomas. However, pathological evaluation of tumor tissues from patients before and after novel treatments is a crucial initial step toward understanding their effects. It also may identify components of tumor morphology that aid in patient selection for anti-angio-genic therapy.

Vasculature and VEGF Expression

In this study, we present our preliminary observations of the effects of Avastin on tumor and blood vessel mor-phology. Tumor cellularity and morphology were simi-lar before and after treatment. Two patients displayed a marked decrease in microvessel density and a striking apparent “normalization” of blood vessels following Avastin therapy, as described by Jain et al.8 After treat-ment with Avastin, bizarre vascular structures such as arcades and glomeruloid proliferations were no longer present in these specimens. Since we did not observe the same phenomena in control cases of high-grade glioma after similar treatment without Avastin, we postulate this may be an effect of antiangiogenic treatment. Unex-pectedly, there was an increase in microvessel counts in one patient who did not have a radiographic response to Avastin. In this case, persistent contrast enhancement on MRI correlated with continuing vascular proliferation histologically.

VEGF immunostains labeled a variable proportion of tumor cells. There was an increase in VEGF immuno-reactivity in one case and a decrease in four cases. The increases in VEGF immunoreactivity do not coincide with an increase with microvessel density nor with a lack of radiographic treatment response. Similarly, no corre-lation is apparent between the percentage of tumor cells labeled prior to therapy and the radiographic response to Avastin treatment.

VEGF also labeled the basement membranes of tumor blood vessels. Vascular basement membranes are well known to play an important role in tumor vascu-

larization: they bind VEGF and may serve as a reser-voir of angiogenic factors.9 In addition, in vivo experi-ments demonstrate that basement membrane sleeves persist after antiangiogenic treatment and serve as a potential scaffold for the sprouting of vascular endothe-lial cells after completion of anti-VEGF treatment.10,11 Such revascularization occurred as early as 7 days after antiangiogenic treatment in mouse models of lung car-cinoma. The intervals between the last Avastin dose and surgical intervention were longer than 1 week in all patients. It is possible that similar revascularization occurred in the interval. Patients 1 and 2 were character-ized by bizarre vascular structures before but not after Avastin therapy. If this is an effect of Avastin, the effect persisted for weeks to months.

The possibility that pretreatment VEGF expression may predict clinical response to antiangiogenic treat-ment should be investigated in larger patient series. In the present study, we did not observe a correlation between pretreatment tumor expression of VEGF and response to bevacizumab, consistent with reports in non-CNS neoplasms. In 169 patients with metastatic colorectal cancer, pretreatment microvessel density and VEGF-A expression on tissue microarrays did not corre-late with effects of Avastin treatment.12 To further study the in vivo actions of Avastin, better characterization of VEGF receptor tyrosine kinase expression patterns and downstream signaling cascades may be useful, as has been done in a variety of tumors with variable results. Bone marrow biopsy specimens from patients with acute myeloid leukemia after treatment with Avastin demon-strated reduced VEGF expression without a correspond-ing change in the expression of phosphorylated activated VEGF receptors.13 In breast cancer patients, Avastin therapy was associated with decreased expression of phosphorylated VEGF receptors without a change in microvessel density or VEGF expression.14

Mesenchymal and Hematopoietic Stem Cell and Invasion Markers

It is interesting to note that three of five patients had multifocal tumor recurrence following Avastin treat-ment. This observation led to a more detailed immuno-histochemical analysis of stem cell, mesenchymal, and tumor invasion markers including CD34, smooth muscle actin, D2-40 or podoplanin, and fascin.

We observed a relative increase in CD34-positive cells following antiangiogenic treatment. CD34 is a glyco-phosphoprotein normally expressed by mature endothe-lial cells, mesenchymal and hematopoetic progenitor cells, and neural stem cells. It is also expressed in CNS lesions associated with epilepsy, such as gangliogliomas, pleomorphic xanthoastrocytomas, and cortical dyspla-sia.15–17 In this context, CD34 immunoreactivity was thought to indicate cellular origin from atypically differ-entiated neural precursors. More recently, expression of CD34 in conjunction with other hematopoietic markers has been reported in a case of medulloblastoma.18 Taken together, this may indicate that expression of such a stem cell marker reflects progression toward a more primi-



tive phenotype. Furthermore, it is possible that CD34-positive cells in gliomas are recruited into the tumor from circulating bone marrow–derived hematopoietic progenitors.19 In vitro experiments demonstrated that bone marrow–derived CD34-positive cells can develop neural and astrocytic phenotypes.20 For future charac-terization of these tumor components, double-staining studies with other stem cell, glial, neural, and endothe-lial markers may be useful.

Smooth muscle actin labels smooth muscle cells or pericytes that form an integral part of microvas-cular proliferations in high-grade gliomas.21 It is also expressed by tumor cells in some glioblastomas and gliosarcomas.22–24 The upregulation of this marker in two of our cases may thus represent a transition to a “mesenchymal” phenotype. Alternatively, there may be a causal relationship between inhibition of VEGF and upregulation of smooth muscle actin: in vitro studies on human CD34-positive vascular progenitor cells demon-strated a differentiation into endothelial cells in response to VEGF, whereas addition of platelet-derived growth factor to the same progenitor cell type produces smooth muscle actin–positive cells with contractile properties.25 It is possible that a blockage of VEGF activity by bevaci-zumab favors the differentiation of vascular progenitor cells toward a smooth muscle cell phenotype.

D2-40 is an immunohistochemical marker recogniz-ing podoplanin, a transmembrane glycoprotein normally expressed in lymphatic endothelium, mesothelium, and glandular myoepithelial cells. It is also expressed in dif-fusely infiltrating astrocytic tumors, and D2-40 expres-sion levels correlate with tumor grade.26,27 Expression is highest in glioblastoma, and thus D2-40 serves as a marker of malignant progression.28 Functionally, podo-planin modifies the actin cytoskeleton and mediates cell migration by filopodia formation.29,30 As a second marker of tumor cell motility, we performed immunos-taining for fascin, an actin-modifying protein impli-cated in formation of cell filopodia, which is abundantly

expressed in many human malignancies, especially high-grade neoplasms.31–37 In gliomas, fascin expression cor-relates with tumor grade and is a putative marker of tumor invasiveness.38,39

While many immunohistochemical markers for glioma invasion are available, we chose these markers for their proven technical reliability and consistency in routine diagnostic pathology. We found the expression of these markers after Avastin was increased in some cases and decreased in others, with similar findings in the control group. We cannot conclude that treatment with Avastin has any effect on the expression of these markers. Of interest, one patient who experienced a dif-fuse recurrence showed robust upregulation of smooth muscle actin, CD34, fascin, and D2-40 after Avastin. In some cases, antiangiogenic treatment may trigger tumor cell migration to areas with robust vascularization. It is also possible that there is a relationship between increased expression of invasion markers and a diffuse recurrence of disease.

Conclusion

This series demonstrates that treatment effects may be observed in situ on surgical specimens. We observed a decrease in microvessel density and a “normalization” of vasculature in some cases, which may be a direct effect of Avastin therapy. We did not observe a corre-lation between expression levels of VEGF, the biologi-cal target of Avastin, and decreased tumor vascularity after treatment. Upregulated expression of hematopoi-etic stem cell, mesenchymal, and invasive markers may be associated with antiangiogenic treatment. Clearly, future studies on larger patient cohorts are necessary for robust clinicopathological correlations to be observed. Such assessment will be essential to validate therapeutic targets and predict treatment response to novel tumor therapies such as Avastin.

References

1. Fischer I, Gagner JP, Law M, Newcomb EW, Zagzag D. Angiogenesis

in gliomas: biology and molecular pathophysiology. Brain Pathol.

2005;15:297–310.

2. Ferrara N. Vascular endothelial growth factor: basic science and clini-

cal progress. Endocr Rev. 2004;25:581–611.

3. Ferrara N. VEGF as a therapeutic target in cancer. Oncology. 2005;

69(suppl):11–16.

4. Ferrara N, Hillan KJ, Gerber HP, Novotny W. Discovery and develop-

ment of bevacizumab, an anti-VEGF antibody for treating cancer. Nat

Rev Drug Discov. 2004;3:391–400.

5. Ferrara N, Hillan KJ, Novotny W. Bevacizumab (Avastin), a humanized

anti-VEGF monoclonal antibody for cancer therapy. Biochem Biophys

Res Commun. 2005;333:328–335.

6. Birner P, Piribauer M, Fischer I, et al. Vascular patterns in glioblastoma

influence clinical outcome and associate with variable expression of

angiogenic proteins: evidence for distinct angiogenic subtypes. Brain

Pathol. 2003;13:133–143.

7. Madconald D, Cairncross G, Stewart D, et al. Phase II study of topote-

can in patients with recurrent malignant glioma. National Clinical Insti-

tute of Canada Clinical Trials Group. Ann. Oncol. 1999;7:205–207.

8. Jain RK, di Tomaso E, Duda DG, Loeffler JS, Sorensen AG, Batchelor

TT. Angiogenesis in brain tumours. Nature Rev Neurosci. 2007;8:

610–622.

9. Sund M, Xie L, Kalluri R. The contribution of vascular basement mem-

branes and extracellular matrix to the mechanics of tumor angiogen-

esis. Apmis. 2004;112:450–462.

10. Inai T, Mancuso M, Hashizume H, et al. Inhibition of vascular endothe-

lial growth factor (VEGF) signaling in cancer causes loss of endothelial

fenestrations, regression of tumor vessels, and appearance of base-

ment membrane ghosts. Am J Pathol. 2004;165:35–52.



11. Mancuso MR, Davis R, Norberg SM, et al. Rapid vascular regrowth

in tumors after reversal of VEGF inhibition. J Clin Invest. 2006;116:

2610–2621.

12. Jubb AM, Hurwitz HI, Bai W, et al. Impact of vascular endothelial

growth factor–A expression, thrombospondin-2 expression, and

microvessel density on the treatment effect of bevacizumab in meta-

static colorectal cancer. J Clin Oncol. 2006;24:217–227.

13. Zahigaric L, Schliemann C, Bieker R, et al. Bevacizumab reduces VEGF

expression in patients with relapsed and refractory acute myeloid

leukemia without clinical antileukemic activity. Leukemia. 2007;21:

1310–1312.

14. Wedam SB, Low JA, Yang SX, et al. Antiangiogenic and antitumor

effects of bevacizumab in patients with inflammatory and locally

advanced breast cancer. J Clin Oncol. 2006;24:769–777.

15. Blumcke I, Giencke K, Wardelmann E, et al. The CD34 epitope is

expressed in neoplastic and malformative lesions associated with

chronic, focal epilepsies. Acta Neuropathol (Berl). 1999;97:481–490.

16. Blumcke I, Lobach M, Wolf HK, Wiestler oD. Evidence for develop-

mental precursor lesions in epilepsy-associated glioneuronal tumors.

Microsc Res Tech. 1999;46:53–58.

17. Reifenberger G, Kaulich K, Wiestler oD, Blumcke I. Expression of the

CD34 antigen in pleomorphic xanthoastrocytomas. Acta Neuropathol

(Berl). 2003;105:358–364.

18. Etzell JE, Keet C, McDonald W, Banerjee A. Medulloblastoma simulat-

ing acute myeloid leukemia: case report with a review of “myeloid

antigen” expression in nonhematopoietic tissues and tumors. J Pedi-

atr Hematol Oncol. 2006;28:703–710.

19. Lyden D, Hattori K, Dias S, et al. Impaired recruitment of bone-

marrow-derived endothelial and hematopoietic precursor cells blocks

tumor angiogenesis and growth. Nat Med. 2001;7:1194–1201.

20. Hao HN, Zhao J, Thomas RL, Parker GC, Lyman WD. Fetal human

hematopoietic stem cells can differentiate sequentially into neural

stem cells and then astrocytes in vitro. J Hematother Stem Cell Res.

2003;12:23–32.

21. Wesseling P, Schlingemann Ro, Rietveld FJ, Link M, Burger PC, Ruiter

DJ. Early and extensive contribution of pericytes/vascular smooth

muscle cells to microvascular proliferation in glioblastoma multiforme:

an immuno-light and immuno-electron microscopic study. J Neuro-

pathol Exp Neurol. 1995;54:304–310.

22. Haddad SF, Moore SA, Schelper RL, Goeken JA. Smooth muscle can

comprise the sarcomatous component of gliosarcomas. J Neuropathol

Exp Neurol 1992;51:493–498.

23. Rangdaeng S, Truong LD. Comparative immunohistochemical staining

for desmin and muscle-specific actin: a study of 576 cases. Am J Clin

Pathol. 1991;96:32–45.

24. Rodriguez FJ, Scheithauer BW, Jenkins R, et al. Gliosarcoma arising in

oligodendroglial tumors (“oligosarcoma”): a clinicopathologic study.

Am J Surg Pathol. 2007;31:351–362.

25. Ferreira LS, Gerecht S, Shieh HF, et al. Vascular progenitor cells iso-

lated from human embryonic stem cells give rise to endothelial and

smooth muscle like cells and form vascular networks in vivo. Circ Res.

2007;101:286–294.

26. Shibahara J, Kashima T, Kikuchi Y, Kunita A, Fukayama M. Podoplanin

is expressed in subsets of tumors of the central nervous system. Vir-

chows Arch. 2006;448:493–499.

27. Takei H, Bhattacharjee MB, Rivera A, Dancer Y, Powell SZ. New immu-

nohistochemical markers in the evaluation of central nervous system

tumors: a review of 7 selected adult and pediatric brain tumors. Arch

Pathol Lab Med. 2007;131:234–241.

28. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T, Matsutani M.

Increased expression of podoplanin in malignant astrocytic tumors as

a novel molecular marker of malignant progression. Acta Neuropathol

(Berl). 2006;111:483–488.

29. Martin-Villar E, Megias D, Castel S, Yurrita MM, Vilaro S, Quinta-

nilla M. Podoplanin binds ERM proteins to activate RhoA and promote

epithelial-mesenchymal transition. J Cell Sci. 2006;119:4541–4553.

30. Wicki A, Lehembre F, Wick N, Hantusch B, Kerjaschki D, Christofori G.

Tumor invasion in the absence of epithelial-mesenchymal transition:

podoplanin-mediated remodeling of the actin cytoskeleton. Cancer

Cell. 2006;9:261–272.

31. Adams JC. Roles of fascin in cell adhesion and motility. Curr Opin Cell

Biol. 2004;16:590–596.

32. Shonukan o, Bagayogo I, McCrea P, Chao M, Hempstead B.

Neurotrophin-induced melanoma cell migration is mediated through

the actin-bundling protein fascin. Oncogene. 2003;22:3616–3623.

33. Vignjevic D, Kojima S, Aratyn Y, Danciu o, Svitkina T, Borisy GG. Role

of fascin in filopodial protrusion. J Cell Biol. 2006;174:863–875.

34. Yamaguchi H, Inoue T, Eguchi T, et al. Fascin overexpression in intra-

ductal papillary mucinous neoplasms (adenomas, borderline neo-

plasms, and carcinomas) of the pancreas, correlated with increased

histological grade. Mod Pathol. 2007;20:552–561.

35. Tsai WC, Chao YC, Sheu LF, Chang JL, Nieh S, Jin JS. overexpression

of fascin-1 in advanced colorectal adenocarcinoma: tissue microarray

analysis of immunostaining scores with clinicopathological param-

eters. Dis Markers. 2007;23:153–160.

36. Jin JS, Yu CP, Sun GH, et al. Increasing expression of fascin in renal cell

carcinoma associated with clinicopathological parameters of aggres-

siveness. Histol Histopathol. 2006;21:1287–1293.

37. Xie JJ, Xu LY, Zhang HH, et al. Role of fascin in the proliferation and

invasiveness of esophageal carcinoma cells. Biochem Biophys Res

Commun. 2005;337:355–362.

38. Peraud A, Mondal S, Hawkins C, Mastronardi M, Bailey K, Rutka JT.

Expression of fascin, an actin-bundling protein, in astrocytomas of

varying grades. Brain Tumor Pathol. 2003;20:53–58.

39. Roma AA, Prayson RA. Fascin expression in 90 patients with glioblas-

toma multiforme. Ann Diagn Pathol. 2005;9:307–311.

High-grade glioma before and after treatment with radiation and Avastin: Initial observations

Documents

Transcript of High-grade glioma before and after treatment with radiation and Avastin: Initial observations