Combining functional imaging and interstitial pressure measurements to evaluate two anti-angiogenic treatments.

BACKGROUND: Interstitial hypertension is responsible for poor capillary blood flow and hampered drug delivery. The efficacy of combined sorafenib/bevacizumab treatment given according to different administration schedules has been evaluated by measuring both interstitial pressure (IP) and quantitative dynamic contrast-enhanced ultrasonography (DCE-US) parameters in melanoma-bearing mice. MATERIAL AND METHODS: [corrected] Sixty mice were xenografted with B16F10 melanoma. Animals received a daily administration over 4 days (D0 to D3) of either sorafenib at 30 mg/kg, bevacizumab at 2.5 mg/kg alone, or different schedules of combined treatments. Perfusion parameters determined using an Aplio® sonograph (Toshiba) with SonoVue® contrast agent (Bracco) were compared to IP measurements using fiberoptic probes (Samba®) at D0, D2, D4, D8. RESULTS: The mean baseline IP values ranged between 6.55 and 31.29 mmHg in all the groups. A transient IP decrease occurred at D2 in all treated groups, and especially in the concomitant group which exhibited a significant IP reduction compared to D0. A significant decrease in both the peak intensity and the area under the curve was observed at D4 in the group with concomitant administration of both molecules which yielded maximal inhibition of the tumor volume and the number of vessels. No correlation was found between IP values and volume or perfusion parameters, indicating complex relationships between IP and vascularization. No IP gradients were found between the center and the periphery but IP values in these two regions were significantly correlated (R = 0.93). CONCLUSION: The results suggest that IP variations could be predictive of vascular changes and that one single IP measurement is sufficient to fully characterize the whole tumor.

Estimation of intra-operator variability in perfusion parameter measurements using DCE-US.

AIM: To investigate intra-operator variability of semi-quantitative perfusion parameters using dynamic contrast-enhanced ultrasonography (DCE-US), following bolus injections of SonoVue(®). METHODS: The in vitro experiments were conducted using three in-house sets up based on pumping a fluid through a phantom placed in a water tank. In the in vivo experiments, B16F10 melanoma cells were xenografted to five nude mice. Both in vitro and in vivo, images were acquired following bolus injections of the ultrasound contrast agent SonoVue(®) (Bracco, Milan, Italy) and using a Toshiba Aplio(®) ultrasound scanner connected to a 2.9-5.8 MHz linear transducer (PZT, PLT 604AT probe) (Toshiba, Japan) allowing harmonic imaging ("Vascular Recognition Imaging") involving linear raw data. A mathematical model based on the dye-dilution theory was developed by the Gustave Roussy Institute, Villejuif, France and used to evaluate seven perfusion parameters from time-intensity curves. Intra-operator variability analyses were based on determining perfusion parameter coefficients of variation (CV). RESULTS: In vitro, different volumes of SonoVue(®) were tested with the three phantoms: intra-operator variability was found to range from 2.33% to 23.72%. In vivo, experiments were performed on tumor tissues and perfusion parameters exhibited values ranging from 1.48% to 29.97%. In addition, the area under the curve (AUC) and the area under the wash-out (AUWO) were two of the parameters of great interest since throughout in vitro and in vivo experiments their variability was lower than 15.79%. CONCLUSION: AUC and AUWO appear to be the most reliable parameters for assessing tumor perfusion using DCE-US as they exhibited the lowest CV values.

Dynamic contrast-enhanced ultrasonography (DCE-US) and anti-angiogenic treatments.

Dynamic contrast-enhanced ultrasonography (DCE-US) is a current functional imaging technique enabling a quantitative assessment of tumor perfusion using raw linear data. DCE-US allows calculating several parameters as slope of wash-in or area under the curve representing, respectively, blood flow or blood volume. Decrease of vascularization can easily be detected in responders after 1 or 2 weeks of anti-angiogenic treatment for gastrointestinal stromal tumors (GIST), renal cell carcinoma (RCC), and hepatocellular carcinoma (HCC) and is correlated with progression-free survival and overall survival in RCC or HCC. DCE-US is supported by the French National Cancer Institute (INCa), which is currently studying the technique in metastatic breast cancer, melanoma, colon cancer, gastrointestinal stromal tumors and renal cell carcinoma, as well as in primary hepatocellular carcinoma, to establish the optimal perfusion parameters and timing for quantitative anticancer efficacy assessments. Currently 479 patients are included in 19 centers and the preliminary results on 400 patients with 1096 DCE-US demonstrated that the area under the curve (AUC) quantified at 1 month could be a robust parameter to predict response at 6 months.

Advanced hepatocellular carcinoma: early evaluation of response to bevacizumab therapy at dynamic contrast-enhanced US with quantification--preliminary results.

PURPOSE: To investigate whether there is any correlation between standard efficacy endpoints-specifically, tumor response, progression-free survival, and overall survival-and tumor perfusion parameters measured by using dynamic contrast material-enhanced ultrasonography (US) in patients with advanced hepatocellular carcinoma (HCC) treated with bevacizumab. MATERIALS AND METHODS: The institutional review board approved the study, and all patients provided written informed consent before their enrollment. Between June 3, 2005, and September 28, 2007, 42 patients (33 men, nine women; median age, 62 years; age range, 23-84 years) participated in this phase II study of single-agent bevacizumab treatment. Tumor response (based on RECIST [response evaluation criteria in solid tumors]) at 2 months was assessed in 37 patients, and progression-free survival and overall survival were assessed in all 42 patients. Dynamic contrast-enhanced US (ie, dynamic US) was performed before treatment (day 0); on days 3, 7, 14, and 60 after treatment; and every 2 months thereafter. Tumor perfusion parameters were estimated quantitatively from contrast material uptake curves constructed from raw linear data. The changes in dynamic US functional parameters between day 0 and the later time points were compared between treatment responders and nonresponders by using nonparametric tests. Given multiple comparisons, P < .001 indicated significance. RESULTS: The percentage decrease in several dynamic US parameters between day 0 and day 3 showed trends toward correlation with (a) tumor response in terms of total area under the time-intensity curve (AUC) (P = .02), AUC during wash in (P = .04), AUC during washout (P = .02), and time to peak intensity (P = .03); (b) progression-free survival in terms of time to peak intensity (P = .028); and (c) overall survival in terms of AUC (P = .002) and AUC during washout (P = .003). CONCLUSION: Dynamic US can be used to quantify dynamic changes in tumor vascularity as early as 3 days after bevacizumab administration in patients with HCC. These early changes in tumor perfusion may be predictive of tumor response at 2 months, progression-free survival, and overall survival, and they may be potential surrogate measures of the effectiveness of antiangiogenic therapy in patients with HCC.

Dynamic contrast-enhanced ultrasonography (DCE-US): a new tool for the early evaluation of antiangiogenic treatment.

Dynamic contrast-enhanced ultrasonography (DCE-US) is a new functional technique enabling a quantitative assessment of solid tumor perfusion using raw linear data. DCE-US allows the calculation of parameters as slope of wash-in or area under the curve (AUC) representing, respectively, blood flow or blood volume. Reduction in tumor vascularization can easily be detected in responders after 1 or 2 weeks and is correlated with progression-free survival and overall survival in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). DCE-US is supported by the French National Cancer Institute (INCa), which is currently studying the technique in metastatic breast cancer, melanoma, colon cancer, gastrointestinal stromal tumors and renal cell carcinoma, as well as in primary hepatocellular carcinoma, to establish the optimal perfusion parameters and timing for quantitative anticancer efficacy assessments. Currently 490 patients are included in 20 centers and the preliminary results on 400 patients with 1,096 DCE-US demonstrated that AUC could be a robust parameter to predict response.

Metastatic renal cell carcinoma treated with sunitinib: early evaluation of treatment response using dynamic contrast-enhanced ultrasonography.

PURPOSE: To determine the utility of dynamic contrast-enhanced ultrasonography (DCE-US) as a prognostic tool for metastatic renal cell carcinoma patients receiving sunitinib and to identify DCE-US parameters that correlate with early treatment response. EXPERIMENTAL DESIGN: Thirty-eight patients received 50 mg/d sunitinib on schedule 4/2 (4 weeks on followed by 2 weeks off treatment). After two cycles, response evaluation criteria in solid tumors were used to classify patients as responders or nonresponders. DCE-US evaluations were done before treatment and at day 15; variations between days 0 and 15 were calculated for seven DCE-US functional parameters and were compared for responders and nonresponders. The correlation between DCE-US parameters and disease-free survival (DFS) and overall survival (OS) was assessed. RESULTS: The ratio between DCE-US examinations at baseline and day 15 significantly correlated with response in five of the seven DCE-US parameters. Two DCE-US parameters (time to peak intensity and slope of the wash-in) were significantly associated with DFS; time to peak intensity was also significantly associated with OS. CONCLUSIONS: DCE-US is a useful tool for predicting the early efficacy of sunitinib in metastatic renal cell carcinoma patients. Robust correlations were observed between functional parameters and classic assessments, including DFS and OS.

[Liver chemoembolization: an update]. / Le point sur la chimioembolisation hépatique.

Percutaneous intraarterial techniques for treatment of liver tumors are continuously under development. New therapeutic methods are available (such as microsphere-encapsulated chemotherapy or yttrium 90 microspheres) and presently under evaluation. These methods enlarge the field of indications, i.e. in metastasis from colorectal cancer. Hepatocellular carcinoma and metastasis from neuroendocrine tumours are the main pathologies favourably responding to "conventional" chemoembolization. Thank to controlled studies and meta-analysis performed along the last ten years, curative-palliative-as well as adjuvant-indications for chemoembolization in hepatocellular carcinoma are now wellestablished.

[Targeted treatments: which imaging?]. / Quelle imagerie pour les thérapies ciblées?

Currently, the evaluation of targeted treatments by functional imaging in oncology is a main goal. Several techniques as Dynamic Contrast Enhanced-MRI, CT-perfusion or Dynamic Contrast Enhanced-US are proposed. The blood flow perfusing the tumor, the blood volume corresponding to the percentage of vessels of total tumor or the diffusion of contrast agent are parameters calculated from the acquisition of time intensity curves during several minutes (TIC). Blood flow, blood volume and mean transit time could be calculated by DCE-US, DCE-MRI and CT-perfusion. But the capillary permeability and the interstitial volume could be evaluated only with DCE-MRI and CT-perfusion because US contrast agent is strictly intravascular. These functional imaging techniques allow predicting earlier the clinical response to targeted treatments before the modification of tumoral volume evaluated according to RECIST criteria.

Benefits of contrast-enhanced sonography for the detection of liver lesions: comparison with histologic findings.

OBJECTIVE: The objective of our study was to compare the usefulness of contrast-enhanced sonography with baseline sonography in detecting malignant liver lesions. SUBJECTS AND METHODS: This prospective study included 116 patients. All patients underwent a preoperative conventional sonography examination followed by sonography after injection of contrast agent combined with the use of perfusion software (vascular recognition imaging or pulse subtraction imaging). Histopathologic analysis was the reference standard used to compare the diagnostic value of baseline sonography versus contrast-enhanced sonography. RESULTS: Eighty-two patients underwent hepatic surgery, 31 did not because of disseminated lesions, and the remaining three patients did not meet inclusion criteria. Three hundred six surgically proven lesions were taken into account for comparison of the two techniques: 147 were detected on baseline sonography and 177 on contrast-enhanced sonography. Histopathologic analysis revealed 233 malignant and 73 benign lesions. Sensitivity and specificity were improved on contrast-enhanced sonography compared with baseline sonography for the detection of malignant lesions: 68.7% versus 58.8% and 67% versus 50.7%, respectively. Contrast-enhanced sonography detected 23 additional malignant lesions that had been seen as lacuna at the portal venous phase and characterized as 19 benign nodules, thus improving the performance of sonography in 13.7% of the cases. CONCLUSION: Contrast injection improved the sensitivity and specificity of baseline sonography and should be performed in routine practice if hepatic surgery is being considered for the management of liver lesions.

Acoustic characterization of a new trisacryl contrast agent. Part I: In vitro study.

The objective of the study was to acoustically characterize trisacryl polymeric microparticles (TMP), which are derived from biocompatible embolic agents. With significant acoustic properties, these polymeric particles could be potentially used as targeted ultrasound contrast agents, directed towards a specific site, with ligands conjugation on the polymeric network surface. In the in vitro study, a pulser/receiver (PRF of 1 kHz), associated to different transducers (5, 10 and 15 MHz), was used to measure the acoustic properties of the TMP inserted in a Couette flow device. Acoustic characterization according to TMP concentration (0.12-15.63 mg/ml), frequency (4.5-17 MHz, defined by each transducer bandwidth), ultrasound pressure (137-378 kPa) and exposure time (0-30 min) was conducted. Particle attenuation was also evaluated according to TMP concentration and emission frequency. Backscattering increased non linearly with concentration and maximum enhancement was of 16.4 dB+/-0.89 dB above 7.8 mg/ml. This parameter was found non-linear with increasing applied pressure and no harmonic oscillation could be noticed. Attenuation reached approximately 1.4 dB/cm at 15 MHz and for the 15.6 mg/ml suspension. The TMP have revealed in vitro ultrasound properties comparable to those observed with known contrast agents, studied in similar in vitro systems. However, such set-ups combined with a rather aqueous suspending medium, have some limitations and further investigations need now to be conducted to approach in vivo conditions in terms of flow and blood environment.

Acoustic characterization of a new trisacryl contrast agent. Part II: Flow phantom study and in vivo quantification.

The biocompatible trisacryl particles (TMP) are made of a cross-linked acrylic copolymer. Their inherent acoustic properties, studied for a contrast agent application, have been previously demonstrated in a in vitro Couette device. To measure their acoustic behaviour under circulating blood conditions, the TMP backscatter enhancement was further evaluated on a home-made flow phantom at different TMP doses (0.12-15.6 mg/ml) suspended in aqueous and blood media, and in nude mice (aorta and B16 grafted melanoma). Integrated backscatter (IB) was measured by spectral analysis of the Doppler signals recorded from an ultrasound system (Aplio) combined with a 12-MHz probe. Doppler phantom experiments revealed a maximal IB of 17+/-0.88 dB and 7.5+/-0.7 dB in aqueous and blood media, respectively. IB measured on mice aorta, in pulsed Doppler mode, confirmed a constant maximal value of 7.29+/-1.72 dB over the first minutes after injection of a 7.8 mg/ml TMP suspension. Following the injection, a 60% enhancement of intratumoral vascularization detection was observed in power Doppler mode. A preliminary histological study revealed inert presence of some TMP in lungs 8 and 16 days after injection. Doppler phantom experiments on whole blood allowed to anticipate the in vivo acoustic behaviour. Both protocols demonstrated TMP effectiveness in significantly increasing Doppler signal intensity and intratumoral vascularization detection. However, it was also shown that blood conditions seemed to shadow the TMP contrast effect, as compared to in vitro observations. These results encourage further investigations on the specific TMP targeting and on their bio-distribution in the different tissues.

[Imaging and angiogenesis: DCE-US (dynamic contrast enhanced-ultrasonography)]. / Imagerie et angiogenèse: la DCE-US (dynamic contrast enhanced-ultrasonography).

The early and functional evaluation of new treatments in oncology is a main goal. At present, technical advances in Doppler ultrasonography allow the detection of neovascularization for superficial and deep malignant tumours in order to evaluate the efficiency of new treatments such as antiangiogenic molecules. Contrast agents injection improves the efficiency of this technique and developments of perfusion softwares optimize this detection. Slow flows in tumour-microvessels can be detected. Treatment response using DCE-US (dynamic contrast enhanced-ultrasonography) can be early predicted based on changes in the vascularization before volume modification calculating perfusion parameters maximal as peak intensity, time to peak intensity, area under the curve, slope coefficient of wash-in.

Methodology for quantifying interactions between perfusion evaluated by DCE-US and hypoxia throughout tumor growth.

The objective was to validate a combination of two new technologies to depict tumor physiology both temporally and spatially with dynamic contrast-enhanced sonography and an oximeter. Human cancer prostate tumors xenografted onto mice were followed for three weeks using dynamic contrast-enhanced ultrasonography (DCE-US) to detect tumor perfusion. Time intensity curves in linear data were quantified on four regions-of-interest (ROI, main tumor section and its anterior, central and posterior intra-tumoral areas) to extract three indices of perfusion. An oxygen sensor was guided by sonography to obtain accurate pO(2) measurements in the three predefined areas of tumors during their development. No impact on tumor growth of subsequent pO(2) probe insertion was detected. Among the four ROIs studied, the local central tumor showed significant perfusion and oxygenation variations throughout the experiment. A correlation was observed between local central tumor perfusion and pO(2), both of them decreasing through time (p = 0.0068; r = 0.66). The methodology which we developed demonstrated the potential of combining DCE-US with direct tissue pO(2) measurements, improving the description of complex intratumoral dynamic behavior.

Dynamic contrast-enhanced ultrasonography (DCE-US) with quantification of tumor perfusion: a new diagnostic tool to evaluate the early effects of antiangiogenic treatment.

Dynamic contrast-enhanced ultrasonography (DCE-US) using the contrast agent Sonovue and vascular recognition imaging software is a novel technique that enables the detection of microvessels and quantitative assessment of solid tumor perfusion using raw linear data. Clinical trials have shown that DCE-US can be used to assess the anticancer efficacy of antiangiogenic treatment, for which conventional efficacy criteria based on size are unsuitable. Reduction in tumor vascularization can easily be detected in responders after 1-2 weeks and is correlated with progression-free survival and overall survival. DCE-US is supported by the French Cancer National Institut. This program is currently studying the technique in metastatic breast cancer, melanoma, colon cancer, gastrointestinal stromal tumor, and renal cell carcinoma, as well as in primary hepatocellular carcinoma, to establish the optimal perfusion parameters and timing for quantitative anticancer efficacy assessments.

Gastrointestinal stromal tumors treated with imatinib: monitoring response with contrast-enhanced sonography.

OBJECTIVE: The purpose of this study was to evaluate contrast-enhanced Doppler sonography with perfusion software as a predictor of early tumor response to imatinib (Glivec) in c-kit-positive gastrointestinal stromal tumors (GISTs). SUBJECTS AND METHODS: Thirty patients (59 tumors) with metastases or a recurrence from a GIST were prospectively included in a single-center imaging trial. Contrast-enhanced Doppler sonography was performed with an Aplio scanner the day before (day-1) starting oral treatment (400 mg) and at days 1, 7, 14, 60, 90, and 6 months, 9 months, and 1 year. The percentage of contrast uptake (Levovist or Sonovue) before treatment and at the different stages of follow-up was evaluated by two radiologists. Digitized quantification was performed using Photoshop software. To define the benchmark standard, all patients were rated as responders or nonresponders at 2 and 6 months by a board consisting of oncologists and radiologists who had all clinical and imaging data at their disposal. Changes in the percentage of contrast uptake at each sonographic examination were compared statistically. RESULTS: A total of 185 examinations were performed. Forty-four lesions in 24 patients were completely evaluated at 2 months, and 29 lesions in 15 patients were completely evaluated at 6 months. Initial contrast uptake at day 1 was predictive of the future response. A strong correlation was found between the decline in tumor contrast uptake at days 7 and 14 and tumor response (p < 10(-4)). CONCLUSION: Contrast-enhanced Doppler sonography is a noninvasive imaging technique that allows the early prediction of tumor response in c-kit-positive GIST treated with Glivec.

To predict progression-free survival and overall survival in metastatic renal cancer treated with sorafenib: pilot study using dynamic contrast-enhanced Doppler ultrasound.

INTRODUCTION: The objective of this study was to evaluate dynamic contrast-enhanced Doppler ultrasound (DCE-US) with perfusion software (Vascular Recognition Imaging) and contrast agent injection as a predictor of tumour response, progression-free survival (PFS) and overall survival (OS). PATIENTS AND METHODS: Thirty patients with a metastatic renal cell carcinoma (RCC) already enrolled in a double-blind randomised study were evaluated. Examinations were performed at baseline, and after 3 and 6 weeks on sorafenib or a placebo in patients with tumour targets that were accessible to DCE-US. RESULTS: A total of 85 examinations were performed, 30 at baseline, 28 at 3 weeks and 27 at 6 weeks. The combination of a decrease in contrast uptake exceeding 10% and stability or a decrease in tumour volume allowed us to discriminate seven good responders and 20 poor responders at 3 weeks. There was a statistically significant difference in PFS (p=10(-4)) and OS (p=10(-4)) between good and poor responders. CONCLUSION: DCE-US is a new noninvasive imaging technique which might be an effective tool for evaluating antiangiogenic drugs in renal cancer.

Combination of HIFU therapy with contrast-enhanced sonography for quantitative assessment of therapeutic efficiency on tumor grafted mice.

The objective was to evaluate treatment efficiency of a new high-intensity focused ultrasound (HIFU) prototype combining a therapeutic transducer with a sonographic probe. The optimal HIFU sequence was defined on ex vivo samples before in vivo evaluation of tumor ablation was performed by perfusion quantification after contrast agent injection. The original feature of this prototype is a 9-MHz sonographic probe in a HIFU device and connected to an Aplio (Toshiba) sonograph. Acoustical power and treatment time were determined on ex vivo livers to generate 1-cm-long lesions. Lesion reproducibility was assessed for the power and treatment time selected. The gap between lesions and HIFU displacement shot procedures were optimized to ablate a 1-cm3 volume. The optimized protocol was applied to five murine tumors in vivo. Tumor ablation was quantified according to (1) contrast uptake (CU) after HIFU using perfusion software (Toshiba) in "vascular recognition imaging" mode and Sonovue (Bracco) contrast agent, and (2) the percentage of necrosis quantified on histologic slides. Ex vivo results: optimized settings, at 442 W/cm2 applied during three cycles (3 s on/5 s off) generated 10 identical elementary lesions measuring 9.78 (+/-0.66) * 2.11 (+/-0.33) mm2. A 4-mm gap between adjacent lesions and a 2-min pause between shot lines were found optimal. In vivo results: 60 % (+/-22) mean reduction in CU after HIFU and tumor necrosis histologically estimated at 58 % (+/-5.7) were quantified for the five animals. The therapeutic potential of this HIFU prototype was demonstrated in vivo through objective quantification of tumor ablation based on CU.

Prognostic value of angiogenesis evaluated with high-frequency and colour Doppler sonography for preoperative assessment of primary cutaneous melanomas: correlation with recurrence after a 5 year follow-up period.

OBJECTIVE: To study the value of high-frequency sonography (HFS) and colour Doppler sonography (CDS) in evaluating the 5 year metastatic potential of primary cutaneous melanomas (CM). MATERIALS AND METHODS: 111 CM were studied before surgical resection and 107 were depicted on HFS. The maximal HFS thickness was measured and compared with the Breslow thickness. A CDS study was performed in each tumour. RESULTS: HFS thickness ranged from 0.26 to 8.0 mm and Breslow thickness from 0.15 to 8.0 mm. HFS and Breslow thickness correlated strongly (r > 0.93). Intratumour vessels were depicted in 43 of the 107 CM, of which 40 were thicker than 2 mm. The median follow-up was 61 months and 27 patients developed relapses. In the univariate analyses, neovascularization visualized with CDS, sonographic thickness and the Breslow thickness were significantly linked to relapses (p < 0.0001), as were lymph node status and ulceration (p = 0.007 and 0.004). CONCLUSION: Vascularization was observed mainly in thick primary melanoma. A median follow-up of 5 years showed the prognostic value of angiogenesis evaluated by CDS.

Doppler US with perfusion software and contrast medium injection in the early evaluation of radiofrequency in breast cancer recurrences: a prospective phase II study.

INTRODUCTION: The aim of this study was to determine the efficacy of Doppler ultrasonography (US) with perfusion software and contrast agent injection (DUPC) during radiofrequency (RF) treatment of local recurrent breast cancer. MATERIALS AND METHODS: Ten patients were included in this monocentric prospective phase II study. DUPC was performed for each patient the day before treatment and immediately after RF in the operating suite. RF ablation was followed by a total mastectomy. The results of DUPC were compared to the histologic analysis of the operative specimen. RESULTS: Before RF, contrast uptake exceeded 70% in 5 lesions and was less than 50% in 5 lesions. Immediately after RF, no vascularization was detected with DUPC in 9 of the 10 lesions. Contrast uptake attaining 30% was depicted in the remaining lesion. At histologic analysis, complete tumour destruction was confirmed in 7 of the 10 operative specimens. CONCLUSION: In this study, DUPC is highly efficient and better adapted for the confirmation of tumour destruction in tumours that are hypervascularised before RF compared to hypovascularised lesions.

In vitro echogenicity characterization of poly[lactide-coglycolide] (plga) microparticles and preliminary in vivo ultrasound enhancement study for ultrasound contrast agent application.

OBJECTIVES: This work includes (1) the characterization of a reproducible poly[lactide-coglycolide] (PLGA) microparticle preparation with an optimial mean diameter and size distribution and (2) the preliminary in vivo ultrasonographic investigation of PLGA microparticles. METHODS: A first series of PLGA microparticle preparations (1 to 15 mum) was acoustically characterized on a hydrodynamic device to select the most appropriate for ultrasound contrast agent application. Preparations of 3-microm microparticles were selected, characterized at different doses, and then injected into 20 melanoma grafted mice for contrast-enhanced power Doppler ultrasonography evaluation. RESULTS: The 3-microm microparticles (3.26-microm mean diameter with 0.41-microm standard deviation) led to in vitro enhancement of 18.3 dB at 0.62 mg/mL. In vivo experiments showed 47% enhancement of intratumoral vascularization detection after PLGA injection, significantly correlated (P < 0.0001) with preinjection intravascularization and tumoral volume. No toxicity was histologically observed. CONCLUSION: The 3-microm PLGA microparticles provided significant enhancement in vitro and in vivo without any toxicity.