Resectable Pancreatic Adenocarcinoma: Is the Enhancement Pattern at Contrast-Enhanced Ultrasonography a Pre-Operative Prognostic Factor?

Ultrasound in Med. & Biol., Vol. 35, No. 12, pp. 1929–1937, 2009 Ó 2009 Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/09/$–see front matter

doi:10.1016/j.ultrasmedbio.2009.06.1100

d

Original Contribution RESECTABLE PANCREATIC ADENOCARCINOMA: IS THE ENHANCEMENT PATTERN AT CONTRAST-ENHANCED ULTRASONOGRAPHY A PRE-OPERATIVE PROGNOSTIC FACTOR? M. D’Onofrio,* G. A. ZAMBONI,* R. MALAGO`,* W. MANTOVANI,y F. PRINCIPE,* A. GALLOTTI,* N. FACCIOLI,* M. FALCONI,z P. CAPELLI,x and R. POZZI MUCELLI* * Department of Radiology, University Hospital G.B. Rossi, Verona, Italy; y Department of Medicine and Public Health, University Hospital G.B. Rossi, Verona, Italy; z Department of Surgery, University Hospital G.B. Rossi, Verona, Italy; and x Department of Pathology, University Hospital G.B. Rossi, Verona, Italy (Received 2 February 2009; revised 16 May 2009; in final form 26 June 2009)

Abstract—The aim of our study was to determine whether the enhancement pattern of pancreatic adenocarcinoma at contrast-enhanced ultrasonography (CEUS) is related to patient prognosis after resection. CEUS of 42 resected adenocarcinomas were retrospectively reviewed. Tumors were divided into two groups: group A 5 poorly vascularized (presence of avascular areas) or group B 5 well vascularized (absence of avascular areas). All lesions were resected and underwent pathological examination assessing tumor differentiation as: undifferentiated (poorly differentiated) or differentiated (moderately and well differentiated). Mean vascular density (MVD) was also evaluated. CEUS enhancement and pathology were correlated (Spearman’s test). Survival was analyzed with the Kaplan-Meier method. Multivariate analysis was performed with the Cox regression model. There were 30 differentiated and 12 undifferentiated adenocarcinomas at pathology. At CEUS, 10 lesions were poorly vascularized, whereas 32 lesions were well vascularized. Positive correlation was observed between CEUS groups and tumoral differentiation (rs 5 0.51; p 5 0.001) and between CEUS and MVD (rs 5 0.74; p , 0.0001). Median survival in patients with group A vascularization at CEUS was significantly lower than in group B (p 5 0.015). Cox proportional hazard model revealed the presence of poorly vascularized tumor at CEUS (p 5 0.0001) as a predictor of higher mortality. In conclusion, CEUS enables accurate depiction of the vascularization of adenocarcinoma, with positive correlation to histology grade and MVD. (E-mail: [email protected]) Ó 2009 Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology. Key Words: Pancreatic adenocarcinoma, Contrast-enhanced ultrasound, Mean vascular density, Prognostic factor, Survival.

Adenocarcinoma represents 80% of malignant pancreatic tumors and its incidence has been increasing in the last decades for unknown reasons (Schima et al. 2007). In more than 95% of the cases, however, independent of its location, pancreatic carcinoma is diagnosed at an advanced stage (Schima et al. 2007). Undifferentiated tumors are more aggressive. Surgical resection of the tumor, with specimen margins microscopically free from neoplastic cells (R0) at pathology, is the only possible curative treatment (Schima et al. 2007). However pancreatic surgery remains hampered by significant morbidity, with a mortality rate of up to 1–5%, even in high-volume referral centers (Alexakis et al. 2004; Tran et al. 2004; Schima et al. 2007). The 5-y survival rate after a R0 resection is estimated at 20–40%, whereas R1 (microscopically positive specimen margins) or R2 (macroscopically positive specimen margins) resections result in lower survival rates (Hermanek 1991; Yeo et al. 1995). Therefore, the

INTRODUCTION The introduction of contrast media has improved the diagnostic capabilities of ultrasound (US). In the last years, the clinical applications of contrast-enhanced US (CEUS) have increased in number. One of the most recent applications of CEUS is the study of pancreatic pathology (Kitano et al. 2004; D’Onofrio et al. 2007b). Among its oncologic applications, CEUS has proven to be highly accurate in depicting tumoral vascularization (Krix et al. 2004, 2005; Murphy-Lavallee et al. 2007; Nagase et al. 2003; D’Onofrio et al. 2005; Numata et al. 2005).

Address correspondence to: Mirko D’Onofrio, Department of Radiology, University Hospital G.B. Rossi, Piazzale L.A. Scuro 10, 37134, University of Verona, Verona, Italy. E-mail: mirko.donofrio@ univr.it 1929

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importance of a correct preoperative staging of pancreatic carcinoma, which is based mainly on imaging techniques, is required. Unfortunately, radiological series report negative predictive values as low as 80% for resectability (Prokesch et al. 2002; Schima et al. 2002). The attention in the development and the interest in the use of biological parameters/markers for a more accurate preoperative evaluation of the malignancy derive from the need of better patient management, which could result by combining biological/functional and morphological/imaging features. Tumoral perfusion is a functional feature of adenocarcinoma resulting from neoangiogenesis and tumor vascularization strictly correlates with tumor histology grade. The purpose of our study was to correlate the enhancement pattern at CEUS of resectable pancreatic carcinoma with pathologic tumor differentiation and patient survival. MATERIALS AND METHODS Our institutional review board approved this retrospective study and waived the need for informed consent. From the surgical registry of pancreatic tumors, all patients who underwent resection of pancreatic adenocarcinoma in our hospital between 2004 and 2006 were retrieved. The period was chosen to warrant a follow-up period of 2 y. Patients Only patients with resectable pancreatic adenocarcinoma who underwent preoperative CEUS in our institution were included in this study. From a total of 148 patients who underwent resection of pancreatic adenocarcinoma, we included 42 patients with pancreatic adenocarcinoma deemed resectable at CT and studied with B-mode US and CEUS before surgery. For the other 106 patients, CEUS was not performed or images were not retrievable. All patients underwent surgery within a week from CEUS and had confirmed pancreatic adenocarcinoma according to histology of the resected specimens. Follow-up was updated yearly, or at shorter intervals if necessary, and ended in 2008. CEUS examination Patients underwent US and CEUS after a 6-h fast. All CEUS examinations were performed by radiologists with .5 y experience and dedicated to pancreatic imaging. Each contrast-enhanced study was performed after a complete B-mode US examination. Examinations were performed on a Sequoia 512 (Acuson, Siemens, Erlangen, Germany) ultrasound system, with contrast-specific US imaging modes with low acoustic ultrasound pressure (2 to 4 MHz coherent contrast imaging or cadence contrast pulse sequencing; mechanical index ,0.2; 12 to 13

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frames/s). The contrast medium used was constituted by sulphur hexafluoride–filled microbubbles with a phospholipids peripheral shell (SonoVueÒ, Bracco, Milan, Italy). A 2.4-mL bolus of contrast medium was hand-injected intravenously, immediately followed by a 5-mL saline flush. Insonation of the pancreatic lesion, along the maximum diameter, was continuous with dynamic observation from the unenhanced phase to the contrastenhanced phases. The arterial phase (~15 to 20 s after injection) was determined when observing maximal hyperechogenicity within the aorta and celiac and mesenteric arteries. The venous phase (approximately 30 to 45 s after injection) is defined when the spleno-mesentericportal tree becomes hyperechoic. The maximum examination time was 5 min. All the video clips were archived on a personal computer via MOD (magneto-optical disk). Qualitative imaging analysis Two radiologists, both blinded to the final pathological results and the preoperative imaging findings, retrospectively reviewed by consensus the US and CEUS examinations, specifically assessed the enhancement pattern in the arterial and venous phase and compared the tumor with the normal adjacent parenchyma and the echogenicity of the lesion in the noncontrast-enhanced phase. A qualitative analysis of the tumor enhancement was performed by judging the grade of enhancement, both in the arterial and venous phase, with respect to the adjacent parenchyma (arbitrarily classified as 3) and by analyzing the presence of at least one avascular intralesional area. Tumor enhancement was graded into four classes: class 1 5 markedly hypoechoic/hypovascular with avascular intralesional areas (MHYPO); class 2 5 hypoechoic/ hypovascular (HYPO); class 3 5 isoechoic/isovascular (ISO); class 4 5 hyperechoic/hypervascular (HYPER). The chosen classification, already described in literature (Kitano et al. 2004), was based on the assumption that ductal adenocarcinoma is a hypovascularized tumor but at CEUS, compared with computed tomography and magnetic resonance imaging, different degrees of hypovascularization can be seen. In particular, during CEUS examination in some hypovascular pancreatic adenocarcinoma, evident avascular areas related to lesional necrosis and/or fibrosis as previously described by Numata et al. (2005) can be detected. The authors assumed that identification of the avascular areas related to necrosis or fibrosis may indicate, preoperatively, a greater aggressiveness and/or an undifferentiated form of the tumor. All lesions were then divided into two main groups: group A 5 poorly and inhomogeneously vascularized lesions with avascular areas (including class 1), and group

Resectable pancreatic adenocarcinoma and CEUS d M. D’ONOFRIO et al.

B 5 homogeneously vascularized lesions (including classes 2, 3 and 4). Quantitative imaging analysis To verify the qualitative data obtained, one operator calculated tumor/tissue ratio for each tumor by means of computed analysis. The video clip was visualized with the ViewPro program (ViewPro Net Beta, Version 2.0, 1996Y1998). The single images were standardized to obtain a normalization of the gain differences in echogenicity introduced during the examination, as previously described (D’Onofrio et al. 2007a). The process was performed using Adobe Photoshop 6.0.1 (Adobe Systems, San Jose, CA, USA). In particular, on each selected image rendered in greyscale, two reference points of maximum and minimum brightness were selected as input values and the image was then standardized with fixed output values (0 and 255). After this operation, the native images from contrast-enhanced studies were standardized to make them comparable. The quantitative analysis of the enhancement was obtained by calculating the greyscale median (GSM; median of the frequency distribution of the grey levels of the pixels) of a region-of-interest (ROI) localized in the tumor and in the adjacent parenchyma. Tumor/tissue ratio was then calculated. Pathologic examination All the resected specimens were examined by a single pathologist with more than 20 y of experience in pancreatic pathology, blinded to the results of CEUS. In our protocol, histological sections were prepared with hematoxylin-eosin stains and with an endothelial immunohistochemical marker (CD34). Histological sections

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Table 1. Main Characteristics of 42 Patients with Pancreatic Adenocarcinoma

Male/Female Age at surgery (y)* Follow up (mo)y Mortality at conclusion of follow-up period Ca 19-9 (U/mL)* CEA (ng/mL)* Neoplasm size (cm)* Neoplasm sited in the head

Total

n 5 42

23/19 64.0 20.0 22 132.0 2.4 2.5 36

54.8%/45.2% (57.0, 72.0) (3, 51.0) 52.4% (53.0, 433.0) (1.9, 5.1) (1.0, 3.0) 85.7%

*Median(interquartile range). yMedian(range).

were examined first with low magnification (20 to 40x), and then at higher magnification (100 to 200x). Pathological grading was performed based on tumor differentiation, and lesions were divided into undifferentiated tumors (including poorly differentiated tumors) and differentiated tumors (including moderately differentiated and well-differentiated tumors). The vessel counts were assessed in the three areas of the tumor, with the highest number of capillaries and small venules, as described by Tanigawa et al. (1996) and Kuwahara et al. (2003), and the same calculation was performed on the pancreatic parenchyma adjacent to the resected lesion to perform a comparison. Lesional vascularization was assigned comparing the number and size of the lesion vessels to that of the perilesional pancreatic parenchyma present in the resected specimen. A mean vascular density value (MVD) in a range from 1 to 4 was assigned to each lesion from the comparison with the adjacent pancreatic parenchyma (arbitrarily classified as 3). Lesions with fewer vessels were assigned to class 2. Lesions with markedly fewer vessels, owing to the

Fig. 1. Ductal adenocarcinoma markedly hypoechoic at CEUS. (a) CEUS study shows a hypoechoic mass of the pancreatic head resulting as markedly hypoechoic (CEUS class 1/MHYPO) with avascular intralesional area (arrow). (b) Final pathologic diagnosis was pancreatic ductal adenocarcinoma markedly hypovascularized (class P1) with avascular area (asterisk) at MVD analysis by using endothelial immunohistochemical marker (CD34).

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presence of avascular intralesional areas, were assigned to class 1. Lesions with more vessels were assigned to class 4. In summary, at pathology the lesions were divided into four classes: P1 5 markedly hypovascular with avascular intralesional areas; P2 5 hypovascular; P3 5 isovascular; and P4 5 hypervascular. All lesions were then divided into two main groups: poorly vascularized lesions (including class P1) and homogeneously vascularized lesions (including classes P2, P3 and P4). Data analysis All demographic, laboratory, CEUS and clinical data were collected on a database used for all pancreatic tumors. Distribution of continuous variables is reported as median and interquartile range (IQR) (25th, 75th percen-

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tiles). Categorical variables are presented as numbers and percentages. The comparison between CEUS groups was performed using Student’s t-test or the MannWhitney U test, for continuous variables. Qualitative data were compared with the chi square test or Fisher’s exact test when necessary. Ordinal-by-ordinal correlation was tested with Spearman’s test, whereas intervalby-interval correlation was tested with Pearson’s test. The main outcome considered was survival, defined as the time from surgical resection to death. Follow-up was updated on May 31, 2008, giving a potential follow-up of 24 months to each survivor. One patient died of surgical complications and was excluded from the survival analysis; no other patient died from causes other than pancreatic cancer. Survival probability was estimated according to the Kaplan-Meier method, whereas the log-rank test (all cases

Fig. 2. Ductal adenocarcinoma isoechoic at CEUS. (a) CEUS study performed with split-screen visualization software showing contrast-enhanced image on the left side and unenhanced image of the same frame on the right side. A small hypoechoic nodule is visible at the pancreatic head (arrow) resulting as isoechoic (CEUS class 3/ISO), no longer visualized, at CEUS. (b) Final pathologic diagnosis was pancreatic ductal adenocarcinoma isovascularized (class P3) at MVD analysis by using endothelial immunohistochemical marker (CD34).

Resectable pancreatic adenocarcinoma and CEUS d M. D’ONOFRIO et al.

weighted equally) and Breslow test (earlier events weighted more heavily) were used for univariate survival analysis in different subgroups. Multivariate analysis was performed with the Cox regression model to evaluate the role of CEUS class 1 (MHYPO) as an adjusted mortality predictor. A p-value , 0.05 was chosen as significant. Statistical analysis was performed by using commercially available software (SPSS, release 11.5; SPSS, Chicago, IL, USA).

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No statistically significant differences were observed regarding patient gender, size and site of the lesion, surgical specimen margins and tumoral biomarkers (Ca19-9 and CEA) between the two groups (Table 2). Quantitative imaging analysis At computed analysis, the tumor/tissue ratios ranged from 0.1–0.3 in the 1/MHYPO class (Fig. 3), from 0.5–0.7 in the 2/HYPO class, from 0.9–1.1 in the 3/ISO class and from 1.2–1.3 in the 4/HYPER class (Table 3).

RESULTS All lesions were single: 36 were located in the head of the pancreas, five in the body and one in the tail. The maximum diameter of the lesions ranged between 1.2 and 4.3 cm (mean 5 2.68 cm; SD 5 0.66). The demographic and clinical characteristics of the 42 patients included in our study population are summarized in Table 1. All 42 lesions were well depicted by US as hypoechoic masses. Qualitative imaging analysis At CEUS, during the arterial and venous phases, 10 lesions were markedly hypovascular (MHYPO) and included in group A (Fig. 1), and 19, 10 and 3 lesions were hypovascular (HYPO), isovascular (ISO) and hypervascular (HYPER), respectively, and homogeneously vascularized, so they were included in group B (Fig. 2). At histology, 30 pancreatic adenocarcinomas were differentiated and 12 undifferentiated. The pathological analysis of tumoral vascularization showed 6 markedly hypovascular lesions, 23 hypovascular lesions, 12 isovascular and 1 hypervascular lesions. Among the 6 markedly hypovascular lesions, 3/6 (50%) had intralesional necrotic areas, 2/6 (33%) fibrotic intralesional areas and 1/6 (17%) intralesional mucinous inclusions.

CEUS and pathology Among the 10/42 lesions poorly vascularized at CEUS, 7/10 (70%) were undifferentiated tumors, and among 32/42 lesions homogeneously vascularized, 27/ 32 (84.4%) proved to be differentiated tumors. CEUS enhancement and pathological results were compared with Spearman’s correlation test: a good correlation was found between CEUS groups and tumoral differentiation (rs 5 0.51; p 5 0.001). A positive and strong correlation was observed between vascularity at CEUS and MVD (rs 5 0.74; p , 0.0001) comparing CEUS (1/MHYPO, 2/HYPO, 3/ ISO, 4/HYPER) and pathologic (P1, P2, P3, P4) classes (Figs. 1 and 2). CEUS and survival rate At the end of follow-up, 21 patients had died of disease: 7 (70%) in group A, 14 (44%) in group B. As noted before, one patient died at 3 mo because of postoperative complications and was excluded from survival analysis. Median follow-up time was 20 mo (interquartile range 14.5 to 24.0). The 41 patients without perioperative mortality showed a first-, second- and third-year cumulative survival rate of 87.1%, 46.5% and 16.5%, respectively (median survival 24 mo; 95% CI 20, 28). Median survival

Table 2. Demographic Characteristics, Pre-operative Data and Pathological Features of CEUS Pattern in 42 Patients with Pancreatic Adenocarcinoma

Age (y)* Male/Female Neoplasm size (cm) Ca 19-9 (U/mL)* CEA (ng/mL)* Neoplasm site (right gland) Positive surgical margin Pathological featuresy Differentiated Undifferentiated

Group A

n 5 10

Group B

n 5 32

p

59.0 3/7 2.2 164.0 3.0 10 3

(51.3, 66.8) 30.0%/70.0% (2.0, 3.0) (130.0, 1240.0) (1.9, 4.7) 100.0% 30%

66.5 20/12 3.0 126.0 2.4 26 11

(58.0, 73.0) 62.5%/37.5% (2.4, 3.3) (44.0, 277.8) (1.7, 5.8) 81.3% 34.4%

0.062 0.143 0.108 0.104 0.846 0.308 0.899

3 7

30.0% 70.0%

27 5

84.4% 15.6%

0.002

Group A: MHYPO 5 markedly hypoechoic/hypovascular. Group B: HYPO 5 hypoechoic/hypovascular; ISO 5 isoechoic/isovascular; HYPER 5 hyperechoic/hypervascular. *Median(interquartile range). ySpearmancorrelation: 0.51, p 5 0.001.

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Fig. 3. Quantitative analysis of the enhancement of a pancreatic head ductal adenocarcinoma markedly hypoechoic at CEUS. (a) Calculation of the greyscale median (GSM) of a ROI localized inside the tumor. (b) Calculation of the greyscale median (GSM) of a ROI localized inside the perilesional pancreatic parenchyma. A tumor/tissue ratio of 0.3 resulted.

was 16 mo in patients within the MHYPO class, 24 mo in the HYPO class, 35 mo in the ISO class and 22 mo in the HYPER class tumors. Univariate survival analysis with Kaplan-Meier function and unadjusted Cox model (summarized in Table 4) showed CEUS pattern class 1/MHYPO (group A) and pathological differentiation features (undifferentiated) as significant determinants of mortality. In fact, median survival in patients with 1/MHYPO CEUS class (group A) tumoral vascularization was significantly lower (p 5 0.015), equal to 16 mo (95% CI 10, 22), than that in patients with 2/HYPO, 3/ISO and 4/HYPER CEUS classes (group B), equal to a median survival of 25 mo (95% CI 18, 32). Analyzing the pathological results, median survival in patients with undifferentiated adenocarcinoma was 17 mo

(95% CI 6, 28) and 24 mo (95% CI 19, 29) in patients with differentiated adenocarcinoma (Breslow test p 5 0.044). The Cox proportional hazard model, adjusted for preoperative variables (gender, age, size and site of neoplasm; Ca19-9 and CEA levels; CEUS pattern), revealed as predictor of higher mortality the presence of CEUS MHYPO pattern (group A): HRMHYPO 2.96 (95% CI 1.17, 7.49); p 5 0.0001 (Fig. 4). DISCUSSION Survival rate after surgery is reported to be influenced by the presence of residual tumor at resection margins (Hermanek 1991; Yeo et al. 1995). A correct preoperative staging of pancreatic carcinoma, currently based mainly on imaging methods, is therefore required.

Resectable pancreatic adenocarcinoma and CEUS d M. D’O NOFRIO et al.

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Table 3. CEUS Class of Enhancement, ROI mean value and Tumor/Tissue Ratio of 42 Pancreatic Ductal Adenocarcinomas ID

CEUS Classes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

ISO MHYPO MHYPO HYPO ISO HYPO HYPO HYPO HYPO ISO ISO HYPO ISO HYPO IPER MHYPO MHYPO HYPO ISO IPER HYPO MHYPO ISO ISO MHYPO MHYPO ISO HYPO ISO IPER ISO HYPO ISO HYPO HYPO HYPO HYPO HYPO HYPO HYPO HYPO ISO

Tumor ROI

SD

Pancreas ROI

SD

Tumor/Tissue Ratio

106.78 26.34 28.05 77.99 175.46 53.55 60.36 66.47 52.47 111.92 91.59 61.64 100.18 87.98 175.61 9.35 41.56 55.53 103.54 94.25 57.08 33.52 69.83 80.56 34.66 13.64 71.2 64.22 83.36 153.9 90.14 80.57 109.63 56.12 57.33 86.04 82.78 76.06 59.52 67.24 65.98 90.93

25.93 4.74 6.47 11.97 31.01 16.83 6.93 15.27 17.64 28.09 15.13 28.24 27.65 20.35 35.62 6.4 7.51 14.31 19.1 20.76 11.63 8.05 11.77 18.23 12.23 8.89 18.64 15.62 14.65 26.46 17.41 24.41 18.22 13.08 12.17 18.67 16.41 21.82 15.27 19.55 24.68 21.81

120.59 82.76 93.01 115.19 171.63 92.64 98.44 89.73 78.36 115.51 92.36 84.43 112.69 124.46 147.4 107.95 123.44 89.23 120.22 72.05 94.56 107.89 80.37 74.46 131.91 70.89 76.67 118.7 93.33 120.87 100.82 109.42 113.5 89.02 91.11 145.66 116.26 110.69 111.96 121.82 121.74 97.34

23.61 18.06 11.14 25.31 30.36 23.16 13.74 22.2 26.2 31.43 17.89 26.57 28.52 21.6 30.3 30.27 25.34 26.37 26.09 28.32 22.71 24.39 13.26 15.28 28.37 29.92 18.45 23.52 21.85 24.37 22.8 27.28 21.72 21.15 16.08 30.37 29.37 31.05 28.51 31.06 22.53 22.37

0.9 0.3 0.3 0.7 1 0.6 0.6 0.7 0.7 1 1 0.7 0.9 0.7 1.2 0.1 0.3 0.6 0.9 1.3 0.6 0.3 0.9 1.1 0.3 0.2 0.9 0.5 0.9 1.3 0.9 0.7 1 0.6 0.6 0.6 0.7 0.7 0.5 0.6 0.5 0.9

Transabdominal contrast-enhanced ultrasonography (CEUS) is a real-time examination performed with a purely blood-pool microbubble contrast agent (EFSUMB Study group 2004, 2008; Galie` et al. 2005; Solbiati et al. 2001). The usefulness of CEUS has been widely assessed in hepatic imaging (EFSUMB Study group 2004, 2008; Quaia et al. 2004; Solbiati et al. 2001), and its applications in the study of the pancreas are increasingly reported in the literature (D’Onofrio et al. 2005, 2007b; Kitano et al. 2004; Nagase et al. 2003; Numata et al. 2005). CEUS has been shown to be accurate in the evaluation of the vascularization of solid pancreatic lesions (D’Onofrio et al. 2007b; Nagase et al. 2003; Numata et al. 2005). In the present study, the majority (70%) of poorly vascularized, class1/MHYPO (group A) lesions at CEUS were undifferentiated tumors at pathology. Thus, the finding of a poorly vascularized adenocarcinoma at CEUS could suggest the undifferentiated grade.

Angiogenesis is a biological process that leads to the creation of new vessels, with a critical role in tumor growth and metastatic spread (Seo et al. 2000) and results in an increase in microvascular density (MVD). Vascular endothelial growth factor (VEGF) expression in adenocarcinoma is reported to be associated with a high microvascular density, a high risk of liver metastases and shorter survival (Seo et al. 2000). Significant correlation was found between the ratio of patent vessels to total vessels and CEUS enhancement (Nagase et al. 2003). In a series of 42 resected pancreatic tumors, a strong correlation (Rs 5 0.914; p , 0.0001) between CEUS enhancement and the number of CD34 positive tumoral vessels at pathology has been reported in the literature (D’Onofrio et al. 2005). Numata et al. (2005) has shown that the reduced vascularization in pancreatic carcinoma is most often a result of the presence of fibrosis and necrosis. A

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Table 4. Univariate Survival Analysis in 41 Patients with Pancreatic Adenocarcinoma Kaplan-Meier Survival Analysis p-value Median (mo) Age (y)* Gender Female Male Ca 19-9 (U/mL)* CEA (ng/mL)* Neoplasm site Body-tail Head Size (mm)* CEUS group Gr_B Gr_A Pathological Features Differentiated Undifferentiated Post-surg. residual R0 R1-2

(95% CI of median)

Log-rank

Unadjusted Cox Analysis Breslow

Not Applicable

Hazard Ratio

(95%CI of Hazard Ratio)

0.97

(0.93, 1.01)

23 35 Not applicable Not applicable

(20, 26) (16, 54)

0.361

0.959

1.00 1.52 1.04 1.00

— (0.61, 3.84) (0.98, 1.09) (0.99, 1.00)

22 24 Not Applicable

(—, —) (20, 28)

0.577

0.773

1.00 1.42 0.56

— (0.41, 4.92) (0.26, 1.23)

25 16

(18, 32) (10, 22)

0.015

0.008

1.00 2.96

— (1.17, 7.49)

24 17

(19, 29) (6, 28)

0.144

0.044

1.00 1.92

— (0.78, 4.72)

25 21

(19, 31) (15, 27)

0.603

0.323

1.00 1.61

— (0.61, 4.23)

Event: dead of disease. One patient with post-operative mortality excluded. Post-surg. Residual 5 surgical parenchyma margin after tumor resection (R0 5 negative; R1 5 microscopically positive; R2 5 macroscopically positive). *Continuous variables (evaluated only with unadjusted Cox model).

reduction in the microvascular density, and in perfusion, can therefore correspond to an increase in tumor aggressiveness. The authors assumed that the identification of avascular areas related to necrosis or fibrosis may indicate preoperatively a greater aggressiveness and/or an undifferentiated form of the tumor. In our series, a positive correlation was observed between CEUS enhancement classes and pathology MVD, resulting in a worse prognosis for patients with markedly hypovascular tumors at CEUS. This confirms the data presented in a series of 35 advanced pancreatic carcinomas, mainly metastatic, in which only two were homogeneously enhanced at CEUS, whereas the majority showed poorly vascularized patterns (Masaki et al. 2005). MVD evaluates the total number of vessels and CEUS depicts the open intratumoral and peritumoral vessels (Galie` et al. 2005). The difference between tumor perfusion and MVD conciliates our results with those of Seo et al. (2000), because a high VEGF expression would correspond to a high MVD but to a low dynamic tumor perfusion at CEUS. Analyzing survival in our series, the median survival of patients with markedly hypovascular tumors was significantly lower, given that no statistically significant difference was found between the two CEUS groups regarding patient gender, size and site of the lesion, postsurgery residual and tumor biomarkers. Chemotherapy before surgery should be preferable in patients with a particularly worse prognosis related to tumor biology, even if the tumor shows resectability at imaging.

The analysis of prognostic factors before treatment may be helpful in selecting appropriate candidates for chemotherapy (Oshikawa et al. 2002) but also for surgery, in the presence of tumor resectability at imaging, thus improving treatment strategies based on tumor prognosis. Markedly hypovascular CEUS pattern, in our series, appears as a useful parameter for preoperative prognostic

Fig. 4. Cumulative survival probability for CEUS group A and group B. Adjusted survival function by CEUS (Cox’s proportional hazards model [adjusted for gender, age, size and site of neoplasm; Ca 19-9 and CEA levels; CEUS pattern]; HRMHYPO 2.96 [95% CI 1.17, 7.49] p 5 0.0001). N 5 41 patients; 1 patient with postoperative mortality excluded. Axes: x 5 time of followup (mo). y 5 cumulative survival rate.

Resectable pancreatic adenocarcinoma and CEUS d M. D’ONOFRIO et al.

stratification. We observed that the markedly hypovascular CEUS pattern and undifferentiation at pathology were significant determinants of mortality, with a median survival in patients with MHYPO significantly lower than in the other groups. This could mean that the identification at CEUS of a markedly hypovascular tumor, even if resectable at imaging, could impose tumoral biopsy for grading. From this study, CEUS appears to be significant in dividing preoperatively the patients with adenocarcinoma in subgroups with different prognosis. In our series, the presence of CEUS MHYPO pattern was a significant predictor of higher mortality (hazard ratio: HR MHYPO 2.96). Computed quantification analysis showed that MHYPO class of enhancement is characterized by the presence of a tumor/tissue ratio ,0.3. Moreover, at quantitative analysis, no overlap between the tumor/tissue ratio ranges of different classes was found. Some limitations to our study must be acknowledged. First, the series includes a relatively small number of patients; therefore, further studies with larger numbers of patients are warranted. Our study was retrospective, so we could not evaluate in how many patients the tumor was not seen at CEUS, because we included only lesions studied at CEUS (selection bias). In conclusion, CEUS can accurately depict the vascularization of pancreatic adenocarcinoma with positive correlation to histology grade and MVD in the present study. Moreover, the enhancement pattern of adenocarcinomas at CEUS could be a preoperative prognostic factor. Acknowledgments—The authors thank John J. Hermans, MD, for help in revising the English form.

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