Patient selection
The researchers retrospectively analyzed the images of patients with testicular tumors who underwent conventional ultrasound, CEUS and microvascular ultrasound in the department of ultrasound and urology of the first affiliated hospital of Nanjing Medical University between January to November 2020. The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University(Nanjing, China) and performed according to the Helsinki Declaration. Informed consent was waived since this was a retrospective study. All the participants gave their informed consent for the publication of their images in an online open-access publication. The enrolled patients registered their personal information before examination. Clinical information and laboratory data such as clinical history, tumor side, swelling and pain were also recorded in detail. According to the immunohistochemical results, the confirmed diagnosis of PTL cases and the corresponding images were enrolled in this study.
US, CEUS and microvascular US examination of testis
Two US doctors with more than 10 years' experience (L.Y. and Y.T.) in testicular ultrasound examined all the testicular masses included in this study. Gray-scale US, color Doppler flow imaging (CDFI) and CEUS imaging were performed using a real-time US device (Philips Healthcare, Eindhoven, the Netherlands) equipped with a 6-15 MHz linear array transducer. Microvascular US image was collected using a real-time US device (Aixplorer, SuperSonic Imagine, Aix-en-Provence, France) equipped with a 4-15 MHz linear array transducer. The CEUS examinations were also performed by the same transducer at a low mechanical index (0.08), focus positioned behind the region of interest after the injection of 2.4mL of the sulfur hexafluoride contrast agent SonoVue (Bracco SpA, Milan, Italy), followed by 10mL of 0.9% saline. When the microvascular US was activated, the transducer was placed on the surface of scrotum without compression or movement.
Sonographic Interpretation
The doctors examined the conventional US images first and focused on homogeneity, size, echogenicity (compared with surrounding normal testicular tissue), boundary, margin, extent of infiltration, calcification and presence of the formerly described “straight vessel [9]” or “linear echogenic strands [19]” pattern of testicular mass. The tumor’s flow grade [20], velocity, resistance index (RI) and exist of “straight vessel pattern” were also recorded during the color Doppler evaluation.
CEUS was performed on the lesions including normal testicular tissue and at least more than 50% of the tumor area. During the CEUS examination, the probe was placed on the skin surface of scrotum without movement, and the dynamic images were observed more than 2 minutes after SonoVue injection. The CEUS evaluation was performed by two doctors with more than 10 years working experience in CEUS (L.Y. and H.J.). Size change was compared to the same section in grey-scale US at the peak time in CEUS. Time-intensity curve (TIC) analysis was performed for evidence of the degree of overall enhancement (microperfusion) within the target lesions. And peak intensity(PI), time to peak(TTP), and rise time of tumor and surrounding normal testicular tissue were recorded. According to TIC results, we classified increased, decreased, or similar enhancement patterns of the mass (compared with the intensity of surrounding testicular tissue or contralateral testis). Boundary (clear or unclear), size change and homogeneity were also assessed in enhancement images. We also focused on presence of the formerly described “ linear nonbranching[15]” in macrovascular pattern of CEUS images.
The microvascular US was assessed for evidence of the degree of overall enhancement (microperfusion) within the target lesions, which was characterized as increased, decreased, or similar to normal parenchyma, and the macrovascular pattern was described as linear nonbranching or random similar to CEUS.
Histopathology and immunophenotype
The enrolled four cases were diagnosed by immunohistochemical pathological results after operation or testicular core needle biopsy. All specimens were fixed with 3.7% neutral formaldehyde solution, routinely dehydrated, embedded in paraffin, 3um thick sections, HE and immunohistochemical staining. Immunohistochemical staining: En Vision two-step method was used, and negative and positive controls were also set up. All primary antibodies (EBER, CD20, PAX5) were purchased from Fuzhou Maixin Biotechnology Co., Ltd., and TBS was used as negative control instead of primary antibody.