Assessing the Malignancy of Hepataocellular Carcinoma in Cirrhosis Using the Hepatocellular Contrast Agent Gd-EOB-DTPA and Correlation with the Cytometric DNA Index

doi:10.4236/ami.2013.31001

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Advances in Molecular Imaging, 2013, 1, 1-7

http://dx.doi.org/10.4236/ami.2013.31001 Published Online January 2013 (http://www.scirp.org/journal/ami)

Assessing the Malignancy of Hepataocellular Carcinoma in

Cirrhosis Using the Hepatocellular Contrast Agent

Gd-EOB-DTPA and Correlation with the Cytometric DNA

Index

R. Röttgen1, S. Kobi1, C. Grieser1, F. Streitparth1, H. Al-Abadi2, A. Duerr2, W. Faber2, J. Pratschke3,

P. Neuhaus2, B. Hamm2, L. Lüdemann4

1Klinik für Radiologie, Charite’ Campus Virchow Augustenburger Platz 1, Berlin, Germany

2Klinik für Allgemein-Visceral- und Transplantationschirurgie, Charite’ Campus Virchow Augustenburger Platz 1, Berlin, Germany

3Klinik für Visceral-, Transplantations- und Thoraxchirurgie, Universitätsklinikum Innsbruck, Innsbruck, Austria

4Abteilung für Strahlenphysik, Universitätsklinikum Essen, Essen, Germany

Email: rainer.rö[email protected], [email protected], christ[email protected], florian streitparth @charite.de,

[email protected], [email protected], wladim [email protected], johann.pratsch [email protected],

[email protected], bernd.hamm@charite.de , lutz.luedemann@u k - e ssen.de

Received November 26, 2012; revised December 27, 2012; accepted January 5, 2013

ABSTRACT

Aim: Investigation whether the signal intensity of hepatocellular carcinoma (HCC) on dynamic magnetic resonance

imaging (MRI) with hepatocellular contrast medium (CM) correlates with the histologic malignancy grade and cy-

tometric DNA index. Material and Methods: MRI at 1.5 T with hepatocellular CM Gd-DTPA-EOB of 63 patients (52

men, 11 women; 64.0 ± 8.5 years) with HCC in cirrhosis. SI of the tumor and liver tissue in sequences (T1, T1fs, T2fs,

T1fs + CM (arterial, venous, late venous phase) were correlated lik e the asymmetry coefficient of the SI in the arterial

and venous phases respective the native and late venous phases of the tumor with the histologic malignancy grade and

DNA index indicating the grade of abnormal DNA steam line respective aneuploid DNA. Results: SI did not signifi-

cantly correlate with the histologic malignancy grade or DNA index for precontrast or postcontrast images. There is a

significant correlation for the asymmetry coefficient of the SI in the arterial and venous phases with the DNA-index (p

values of 0.00089 (Pearson) and 0.0082 (Spearman). Conclusion: A highly arterialized hepatocellular carcinoma with

rapid washout in the venous phase corresponds to a higher malignant potential. These findings suggest that the MR pa-

rameters investigated here may predict the malignant potential and prognosis of HCC before surgery.

Keywords: Hepatocellular Carcinoma; DNA Index; DNA Cytometry; Malignancy Grading

1. Introduction

Magnetic resonance imaging (MRI) with contrast me-

dium is currently the best imaging modality for the de-

tection and characterization of hepatocellular carcinoma

(HCC) [1,2]. It is based on the signal enhancement pat-

tern after administration of contrast medium, particularly

extracellular or tissue-specific hepatobiliary agents [2].

Gd-EOB-DTPA (Primovist®, Bayer Healthcare, Berlin,

Germany) is an MR contrast agent combining the proper-

ties of extracellular and hepatocellular agents. This

means that images can be acquired both in the dynamic

phase when the contrast agent is still in the intravascular

space and in the hepatobiliary phase when the agent is

metabolized by the hepatocytes. Accumulation in heap-

tocytes occurs within a few minutes of injection, ena-

bling clear delineation of a HCC from healthy liver tis-

sue.

Dynamic MRI with a fast T1-weighted gradient echo

sequence obtained during the arterial (Figure 1), portal

venous (Figure 2), and late venous phases (Figure 3)

after intravenous bolus administration of the contrast

medium has been shown to be useful for characterizing

HCC, which typically exhibits early arterial enhancement

because it is highly arterialized [3]. Accumulation of

contrast medium indicates a tumor of hepatocellular ori-

gin. In the routine clinical setting, however, differentia-

tion of HCC from regenerative nodules in the cirrhotic

liver continues to pose a challenge. In general, though,

the use of morphologic imaging criteria allows adequate

characterization of an intrahepatic tumor, and histologic

confirmation of the diagnosis of HCC is generally not

necessary before surgery.

R. RÖTTGEN ET AL.

Figure 1. Asymmetry coefficient ACart-ven is plotted against

the DNA index. Different symbols indicate the different

tumor grades: filled black circle—grade 1; open square—

grade 2; filled black square—grade 3; asterisk—grade X.

The solid line represents the linear fit of the correlation

between the two parameters. The result of the linear fit is

described by the following equation: AC art-ven = −0.158 +

0.266*DNA index.

Figure 2. Asymmetry coefficient ACpre-late venous is plotted

against the DNA index. Different symbols indicate the dif-

ferent tumor grades: filled black circle—grade 1; open

square—grade 2; filled black square—grade 3; asterisk—

grade X. There is no correlation between the two parame-

ters.

Accurate diagnostic characterization and differentia-

tion of a malignant liver tumor is done using histologic

grading or DNA cytometry. The latter may have impor-

tant implications fo r treatment as it enables objectiv e and

reliable grading of the malignant potential. Photometric

DNA measurement is based on the quantitative determi-

nation of the DNA content of cell nuclei [4]. The mate-

rial for DNA analysis can be obtained by fine needle

aspiration and may provide important additional infor-

mation on the biologic behavior of a tumor that may alter

the therapeutic strategy and determines the prognosis.

Tumor cells are characterized by numerical and/or struc-

tural chromosome aberrations. Severe chromosomal ab-

erration identified as significant by DNA cytometry is

Figure 3. HCC in liver segment 4 during the arterial phase;

fat-saturated T1-weighted image acquired approx. 20 s af-

ter the start of intravenous contrast medium administration.

There is increased peripheral enhancement suggesting high

arterial perfusion of the lesion.

referred to as DNA aneuploidy and is an indicator of the

malignant potential of a tumor. Hence, DNA ploidy or

the DNA index has been identified to be an important

prognostic indicator in patients who underwent liver

transplantation because of HCC in cirrhosis [4-13].

Jonas et al. showed DNA ploidy to be a useful prog-

nostic factor in patients who received a liver transplant

for HCC in cirrhosis, demonstrating that its predictive

value is superior to tumor staging. In this study, ane-

uploidy, or a high DNA index, indicated a poorer prog-

nosis in liver transplant recipients than diploid and poly-

ploid carcinomas with a low DNA index [14].

The purpose of the study is to evaluate, if an assessing

of the malignancy of hepataocellular aarcinomas in cir-

rhosis using MRI with hepatocellular contrast agent will

be possible.

2. Patients and Methods

Sixty-three patients with histologically proofed HCC in

cirrhosis who underwent MRI of the liver with the heap-

tocellular MR contrast agent Gd-EOB-DTPA were ret-

rospectively included in the study. All of them underwent

resection or liver transplant. The patients had a mean age

of 63.0 ± 10.0 years (5 2 men, 64 .0 ± 8.5 year s, range 43 -

84; 11 women, 58.3 ± 14.9 years, range 32 - 82). Patho-

histologic grading of the patients included in the study

yielded 26 well differentiated (G1), 28 moderately dif-

ferentiated (G2), and 8 poorly differentiated HCCs (G3).

In one HCC, the degree of differentiation could not be

determined (GX).

3. DNA Cytometry

DNA cytometry was available for 52 HCCs.It is meas-

ured by up to 300 tumor cell nuclei, sometimes more,

and 30 located in the same preparation as lymphocytes

and leukocytes were used as analytical reference cells.

R. RÖTTGEN ET AL. 3

The DNA content determined by means of these nuclei

singlecellabsorptionphotometry. The measurement was

carried out in the fine-scanning method using a micro-

scope cytophotometer (Ahrens Medical Supply Tecnic,

argteheide, Germany). The control of the scanning table,

the measuring process and the registration of the absorp-

tion programmatically were effected by process com-

puter. The measured data of the respective total extink-

tion were printed along with the mean, standard deviation,

variance and coefficient of variation through a telephoto

type. The measurement of the integrated optical density

of nuclei is coupled interactive on the monitor of a con-

ventional microscope with a PC-based TV image analy-

sis system. The microscope is equipped with a color TV

camera and appropriate interference filters. Within the

relevant cell population of at least 300 cell nuclei are

measured randomly assigned. The measurement is done

automatically by clicking the relevant nuclei with a

mouse on the screen. A DNA histogram. determined per

measurement DNA content of about 300 cell nuclei is

combined in a cytophotogramm and as an absolute value

in AU (arbitraryunits) specified. The number of cyto-

morphometric nuclei measured is denoted by “n”. The

determination of the 2 c value, i.e., of the DNA-content

corresponding to a normal diploid set of chromosomes

occurs in lymphocytes or leukocytes as a calibration dip-

loid cell population. The so-called standard cells (stan-

dard calibration cell population) have to be set in the

same manner as the measured nuclei of the hepatocytes

Hepatozytenkerne, hydrolyzed and stained according to

Feulgen. The doubling of the 2 c-values is given in the

cytophotogram with 4 c and corresponds to a tetraplo id, a

further doubling (quadrupling) a oktaploiden (8 c) set of

chromosomes. The diagnosis of the DNA histograms are

qualitatively according to the ESCAP Consensus Report

in the categories of DNA diploid, DNA polyploid and

DNA-aneuploid and is performed according to the fol-

lowing criteria: diploid DNA: DNA base line (STL) >

1.80 c < 2.20 c polyploid DNA: DNA base line (STL) >

1.80 c < 2.20 and c > 3.60 c < 4.40 c.DNA aneuploid:

abnormal DNA stem line (STL) < 1.80 c > 2.20, or c <

3.60 c > 4.40 c and/or values > 9 c.

4. MRI-Examination

MRI examinations were performed on a 1.5-Tesla Gene-

sis Signa MR scanner (General Electric Medical Systems,

Wisconsin, USA) using an 8-channel surface receive

only coil. The following pulse sequences were analyzed:

 Native breath-hold T1-weighted 2D gradient echo

(GRE) sequence (fast spoiled gradient echo, FSGE);

repetition time (TR): 212 ms, echo time (TE): 4.6 ms,

flip angle (FA): 80˚, slice thickness (SL): 8 mm, in-

terslice gap: 0 mm, field of view (FOV): 36 × 36 cm,

image matrix: 512 × 512, acquisition rate: 24 slices in

15 s.

 Breath-hold axial T1-weighted 3D GRE sequence

with fat saturation (FS); TR: 119 ms, TE: 6.9 ms, FA:

70˚, SL: 8 mm, FOV: 36 × 36 cm, image matrix: 512

x 512, acquisition rate: 24 slices in 40 s.

 2D fast T2-weighted spin echo sequence (fast/turbo

spin echo, FSE) with FS; TR: 2100 ms, TE: 90 ms,

FA: 90˚, turbo factor, ETL: 21, axial, SL: 8 mm, gap:

0 mm, FOV: 36 cm × 36 cm, image matrix: 512 ×

512, acquisition rate: 28 slices in 1 min 9 s.

 Dynamic breath-hold axial 3D T1-weighted sequence;

arterial and venous phase (2 phases) after manual IV

bolus administration of 0.025 mmol/kg of Gd-EOB-

DTPA (0.1 ml). TR: 10.3 ms, TE: 5.2 ms, FA =15˚,

ETL: 59, SL: 5 mm, gap: 0 mm, FOV: 36 × 36 cm,

image matrix: 256 × 256, acquisition rate: 2 3D data-

sets with 40 slices in 20 s per phase. The arterial

phase was acquired 20 s after contrast medium ad-

ministration, the venous phase 50 s after administra-

tion.

 2D breath-hold T1-weighted GRE sequence (FSGE)

approx. 20 min after manual IV bolus administration

of 0.025 mmol/kg of Gd-EOB-DTPA (0.1 ml). TR:

212 ms, TE: 4.6 ms, FA: 80˚, SL: 8mm, gap: 0 mm,

FOV: 36 × 36 cm, image matrix: 512 × 512, FOV:

256 × 205, acquisition rate: 24 slices in 15 s.

The MRI datasets were analyzed on a workstation

(General Electrics picture archiving and communication

system—GE-PACS, GE Healthcare, USA, Software Cen-

tricity RA 1000, version 2027435-091 using two EIZO

RadiForce GS 220 monitors, gray-scales, EIZO Nanao,

Hakui Corporation, Japan). Signal intensities (SI) with

standard deviations (SD) were measured in regions of

interest (ROIs) placed in the HCC and in representable

noncancerous liver tissue on the standardized pulse se-

quences described above. SI in the tumor ROI was

measured over the entire tumor area visualized in the

slice with the largest axial tumor extent and in an area of

approx. 40 mm2 in the liver tissue.

For standardization of the measured SIs, ratios of SI in

the HCC and noncancerous liver tissue were calculated,

referred to as standardized signal intensity (I) below.







tumor

liver

I= SI

Dynamic contrast enhancement was calculated using

the asymmetry coefficient ACart-ven, which describes the

difference in standardized SIs between the arterial and

venous phase.





 



art-ven

art ven

0.5 artven

SI SI

AC =SI +SI



The asymmetry coefficient AClate-ven describes the dif-

ference in standardized SIs before contrast medium ad-

R. RÖTTGEN ET AL.

ministration and the late venous phase (20 min after IV

contrast medium administration).

 



pre-late-ven

pre

0.5 pr

AC post

e post

I+I



The fact that only these two asymmetry coefficients

could be calculated is due to the pulse sequences that

were available for analysis: Postcontrast images in the

arterial and venous phase were acquired with the T1-

weighted 3D sequence, while nonenhanced images and

the late venous phase were acquired using the T1-

weighted 2D sequence. This is why no asymmetry coef-

ficients could be calculated for nonenhanced SI and the

arterial phase or for the arterial or venous phase and the

late venous phase.

The standardized signal intensities, I, of HCCs deter-

mined from the above-described sequences and the two

asymmetry coefficients (ACart-ven, AClate-ven) were corre-

lated with histopathologic grades in all 63 patients and

with the cytometric DNA index in 52 patients.

5. Results

Analysis by degree of HCC differentiation did not reveal

any significant correlations for the standardized signal

intensities on the unenhanced pulse sequences and the

cytometric DNA index for any of the groups. The same

was true for the standardized signal intensities measured

on the contrast-enhanced images and their correlation

with the DNA indices for any of the pathohistologic

HCC grades.

With increasing DNA indices, we saw a tendency for

higher standardized sign al intensities in the arterial ph ase

and for lower standardized signal intensities in the ve-

nous phase.

In contrast, significant correlations were found for

ACart-ven and the DNA index using both Pearson’s corre-

lation analysis (linear) (p = 0.00089) and the Spearman

rho (nonlinear) (p = 0.0082). The second asymmetry in-

dex investigated, ACpre-late-ven, did not significantly corre-

late with the DNA index (p = 0.67; p = 0.40). No sig-

nificant correlation of the asymmetry index with the tu-

mor grade was found.

The Figures 4 and 5 illustrate the relationsh ips between

the two asymmetry coefficients, ACart-ven and ACpre-late-veno us,

and the DNA index.

6. Discussion

We found no correlation between the MR signal inten-

sities of HCCs on the pulse sequences investigated and

the tumor stage or cytometric DNA index for nonen-

hanced images or the arterial, venous, or late venous

phases of the dynamic series; however, we saw a ten-

dency for increasing DNA indices to be associated with

Figure 4. HCC in liver segment 4 during the venous phase;

fat-saturated T1-weighted image acquired approx. 50 s af-

ter the start of intravenous contrast medium administration.

The signal intensity of the lesion is lower than that of sur-

rounding liver tissue, which indicates rapid washout/reflux

in the venous phase.

Figure 5. HCC in liver segment 4 during the venous phase;

fat-saturated T1-weighted image acquired approx. 50 s af-

ter the start of intravenous contrast medium administration.

The signal intensity of the lesion is lower than that of sur-

rounding liver tissue, which indicates rapid washout/reflux

in the venous phase.

higher standardized sign al intensities in the arterial ph ase

and lower intensities in the ven ous phase. In contrast, we

found a significant correlation between asymmetry coef-

ficient ACart-ven and the DNA index but not for the second

asymmetry coefficient we investigated, i.e., the one be-

tween signal intensities on unenh anced images and those

measured in the late venous phase. These results suggest

that the malignant potential of HCC, expressed in the

DNA index, increases with an increasing difference in

standardized signal intensities between the arterial and

venous phases, as indicated by increasing values of

asymmetry coefficient ACart-ven.

In terms of function, this means that strong arterialize-

tion of a tumor with rapid washout in the venous phase

corresponds to a higher DNA index and hence a higher

malignant potential. Conversely, a lower value for ACart-ven

corresponds to a lower DNA index or malignant poten-

R. RÖTTGEN ET AL. 5

tial.

We attribute this result to the assumption that a high

malignant potential, as indicated by a high DNA index, is

associated with enhanced angiogenesis and results in

tumors with a larger number of arteries and veins. This

assumption would explain the stronger arterial and

weaker venous enhancement we saw in tumors with a

high DNA index, as opposed to tho se with a lower DNA

index. The presence of more vessels in a tumor means

that contrast medium inflow and outflow is both faster

and more pronounced.

Conversely, our initial hypothesis that contrast-en-

hanced MRI using Gd-EOB-DTPA would also improve

the characterization of HCC in cirrhosis with regard to

histopathological tumor grades was not confirmed here.

We found no statistically significant associations be-

tween standardized signal intensities in the arterial, ve-

nous and late phases of the dynamic contrast-enhanced

series and the histopathologic tumor grade. This disap-

pointing outcome might also in part be due to the fact

that HCC is a heterogeneous group with mixed types and

overlap between the different histological grades, making

it difficult at times to assign a tumor to a specific his-

tologic grade .

Based on the results presented here, the dynamic con-

trast behavior of HCC might supplement preoperative

MRI, providing useful diagnostic information that goes

beyond the Milan criteria in evaluating candidates for

liver transplant. Enhancement patterns might provide

clues about the malig na nt poten tial and th e progno sis that

cannot be derived from merely evaluating the morphol-

ogic imaging appearance of HCC. It might also be specu-

lated that our results on the potential role of con-trast

enhancement also apply to other, nonspecific contrast

agents.

Imaging detection of HCC in cirrhosis has been invest-

tigated in many studies [1,15-20]. Schneider et al. and

Hentrich et al. demonstrated the significance of con-

trast-enhanced dynamic MRI using the hepatobiliary

contrast agent Gd-BOPTA (Multi-Hance®) for the de-

tection and differentiation of benign and malignant he-

patic lesions [21]. Hentrich et al. showed that late en-

hancement is related to cellular differentiation with mod-

erately differentiated HCC exhibiting significantly stronger

uptake of contrast medium than poorly differentiated

HCC. Horigome et al. compared MRI including dynamic

contrast-enhanced imaging using Gd-DTPA (Magne-

vist®) with the histopathologic grades of tumor different-

tiation and showed that detection by MRI improves with

poorer differentiation and larger tumor size [22]. Jonas et

al. investigating patients who received a liver transplant

for HCC identified DNA ploidy as a valuable prognostic

factor that is superior to tumor stage and other prognostic

factors. In this study of patients who received a liver

transplant because of HCC, aneuploidy indicated a poor-

er prognosis compared with diploid or polyploid HCCs

[23]. In a univariate analysis, the same study group also

identified the cytometric DNA index as a prognostic pa-

rameter in liver transplant recipients after HCC in cirrho-

sis in addition to the Milan criteria, the histopathologic

tumor grade, vascular invasion, and the cytometric DNA

index. A multivariate analysis identified the DNA index

and vascular invasion as prognostic parameters [10].

The Milan criteria, which are used as a basis for se-

lecting patients with HCC in cirrhosis for liver trans-

plantation, take into account the number and size of le-

sions. In addition, information on differentiation and ma-

lignancy of an HCC would be also useful. Liver trans-

plant recipients meeting the Milan criteria have a mean

5-year survival rate of 62% to 70% when the degree of

differentiation of HCC is not taken into account. Patients

not meeting these criteria have poorer survival rates.

Several histopathologic studies of explanted livers re-

vealed microvascular tumor invasion as an independent

negative predictor of survival after liver transplan t. These

studies also showed a significantly higher incidence of

microvascular invasion with increasing tumor size

[11,24,25]. The risk of microvascular invasion is greater

than 50% for patients with HCC > 5 cm.

Other factors, apart from the Milan criteria, that have

been found to influence survival are the histopathologic

tumor grade, vascular invasion, and DNA cytometry

(DNA ploidy, DNA index) [10,11,23,25]. The signify-

cant association we found between the arterial-venous

asymmetry coefficient, i.e., the ratio of contrast enhance-

ment in the arterial phase to that in the venous phase, and

the DNA index suggests that a dynamic MRI study of the

liver using the hepatocellular contrast agent Gd-EOB-

DTPA provides information on malignancy or the bio-

logical aggressiveness of HCC. Used in patients with

HCC in cirrhosis, this asymmetry coefficient might pro-

vide supplementary information for selecting the sur-

gical procedure (extent of resection/liver transplant) or

serve as a progno s tic factor.

Additional studies—ideally in larger patient popula-

tions—are needed to confirm our findings. The Milan

criteria might then be supplemented by the preopera-

tively determined MRI criterion of the asymmetry coef-

ficient describing the ratio of arterial enhancement to

venous washout following administration of a hepato-

cytic contrast agent.

7. Summary

Our study investigating signal intensities and asymmetry

coefficients determined from preoperative MR images

obtained using the hepatocellular contrast agent Gd-

EOB-DTPA in patients with hepatocellular carcinoma

and cirrhosis revealed a statistically significant relation-

R. RÖTTGEN ET AL.

ship between the asymmetry coefficient of signal en-

hancement in the arterial and venous phases and the can-

cer’s malignant potential as reflected in the DNA index.

The asymmetry coefficient thus has the potential to en-

able preoperative assessment of the malignant potential

of HCC and its prognosis. Preoperative assessment of the

malignancy or aggressiveness of HCC using this MRI

asymmetry coefficient might be a useful supplement to

other imaging criteria such as lesion size, number, and

site.

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