Patients
Our multicenter diagnostic accuracy test study was carried on 175 patients (110 men and 65 women, aged between 13 and 86 years) with pathologically proved, clinically, laboratory, or radiologically suspected metastatic lesions of unknown primary site; they were referred for localization of primary tumor site.
Inclusion criteria
Inclusion criteria were as follows: patients having at least one biopsied metastatic lesion, patients with radiologically suspected metastatic lesion(s), and/or elevated tumor markers.
Exclusion criteria
Exclusion criteria were as follows: uncontrolled diabetes, allergy to intravenous contrast, pregnancy, inability to cooperate with the scan process (inability to lie relatively still for 1–2 h and to lie supine for 30–60 min).
Data collection and analysis were done with approval of the research ethics committee, and written informed consent was obtained from each patient after the nature of the procedures had been fully explained.
FDG PET/CT study
Combined PET/CT scan was performed using Biograph True Point 64 and Biograph Sensation 16 PET-CT (Siemens Medical Healthcare, Erlangen, Germany). The integrated CT system is a 64- and 16-multi-slice scanner. The acquisition of co-registered CT and PET images were performed in one session. Adequate patient preparation rules were strictly followed. Patients were instructed to fast except for glucose-free hydration for 4–6 h before injection of 18F-FDG. The scan was performed 45–60 min (average 55 min) after IV injection of 0.1 millicurie/kilogram [mCi/kg] (3.7–4.5 MBq/kg body weight) with maximum dose of 18 mCi/kg of 18F-FDG. The blood glucose levels were checked in all patients before FDG injection, and no patients showed a blood glucose level of more than 140 mg/dl. The patients were examined in supine position. A whole-body examination was performed starting from skull vault to the feet.
The PET component of the combined imaging system had an axial view of 16.2 cm (per bed position) with an interslice spacing of 3.75 mm in one bed position. The transaxial field of view and pixel size of the PET images reconstructed for fusion were 58.5 cm and 4.57 mm, respectively, with a matrix size of 128 × 128 and 4.5 mm spatial resolution. To avoid artifacts caused by the urinary tract, patients were asked to drink 500 ml tap water or sugar-free green tea 30–60 min prior to image acquisition, and to void just before the start of acquisition. This is to ensure a negative oral contrast within the small bowel and to promote 18F-FDG excretion from the kidneys. No urinary bladder catheterization was used.
A PET scan was performed with several bed positions (12 to 14) depending on the height of the patient, with an axial field of view of approximately 15 cm per bed position with an in-plane spatial resolution of 4 mm covering the same field of view as with CT. The acquisition time of emission data was 2 min per bed position in the three-dimensional mode. The total examination time ranges between 24 and 28 min. Our protocol was adequately satisfied the minimal imaging requirement even though 2 min per bed position is a short acquisition time, relying upon enough injection dose and high sensitivity of Siemens PET camera. The PET section thickness was 3.4 mm. The images were loaded onto a workstation, and attenuation corrections were performed using the CT data. Attenuation-corrected PET images were reconstructed with an ordered-subset expectation maximization iterative reconstruction algorithm.
According to previous recommendation guidelines; CECT data as part of the combined PET/CT examination provide additional information and support lesion detection and characterization. Furthermore, CT contrast agents are of additional value in 18F-FDG PET-negative tumors, so CE PET/CT, even if it involves extra cost, can provide fully diagnostic morphologic and functional data in a single session that could change the clinical management plan and rendering additional diagnostic CT unnecessary [10,11,12].
Thus, a fully diagnostic CT scan was done using the following parameters: 350 mA, 120 kV, 0.5-s tube rotation time, slice thickness 5 mm, 8 mm table feed and 3 mm incremental reconstruction. IV contrast administration of 120 mL of a low-osmolarity iodinated contrast agent (Ultravist 300®, Bayer, Germany) and negative oral contrast agent (water) for bowel was used.
PET images and CT images were fused, displayed, reconstructed, and viewed on workstations (Syngo Multimodality Workplace, Siemens Medical Solutions, AZE Virtual Place Version 3.0035; Azemoto, Tokyo, Japan), which provided multi-planar reformatted PET, CT, and fused PET/CT images with linked cursors as well as maximum intensity projection (MIP) PET images in video mode.
Data interpretation and image analysis
In women of reproductive age, FDG PET/CT imaging was done within a week before or a few days after the menstrual flow phase to avoid any misinterpretation of pelvic FDG PET/CT images [13]. PET/CT images were interpreted in consensus by experienced dual board-certified radiologist/nuclear medicine consultants, one dual board-certified radiologist/nuclear medicine specialist, and one consultant radiologist.
All images were qualitatively and quantitatively interpreted by three dual-qualified consultant radiologists with more than 20 years experience and one dual-qualified specialist radiologist with more than 5 years experience. The presence of abnormal FDG uptake was indicated when accumulation of the tracer was moderately to markedly increased compared to the uptake in normal structures or surrounding tissue, visual in all three planes with the same co-ordinates (x, y, z), with the exclusion of physiological bowel, vessel, and urinary activity. The criterion for malignancy was [18F] FDG hypermetabolism at the site of pathological changes on CT or marked focal hypermetabolism at sites suggestive of malignancy despite absence of signs of pathology at those sites on CT. The distribution of pathological lesions, a prior knowledge of the pattern of spread of different tumors, and the patient’s history were taken into consideration. Quantitative evaluation using standard uptake value (SUV) according to this formula: SUV = (μCi/gram in tissue)/(total μCi injected) body weight. Max. SUV value of more than 3 was considered significant as a reliable predictive value for predicting malignancy. This method of PET/CT SUVmax was selected as a relative minimum cut-off value for best optimal sensitivity and accuracy and was relied upon the average SUVmax results of the previous studies [14,15,16].
During the statistical analysis, patients were categorized based upon the PET/CT results and the final diagnosis. PET/CT results were compared with the final diagnosis. According to the diagnosis of the primary site of malignancy, “primary detected” was classified as true positive (TP) only when it was confirmed histologically during the follow-up. If the finding was confirmed as benign, or if the patient was without any signs of malignancy during the follow-up, the diagnosis was classified as false positive (FP). “Primary unknown” means an evaluation that was classified as true negative (TN) if neither FDG PET nor histological findings or clinical follow-up (including subsequent imaging tests) determined the site of the primary. When the site of the primary was not identified by FDG PET, but was proven histologically or by follow-up using other imaging studies, the finding was classified as being false negative (FN).
Clinical, surgical, and histopathologic findings and correlative imaging modalities were used to assess the results of FDG PET/CT. All detected primary malignancies were hypermetabolic on PET, for all such cases, the final diagnosis was obtained from the medical records, including pathologic reports by biopsy or operation as well as clinical/radiological follow-up. The data gathered during the histopathological examination and clinical follow-up was considered as the reference standard and defined as the final diagnosis. Ultrasonography, mammography, bronchoscopy, endoscopy-colonoscopy, and biopsy were performed as diagnostic tests for patients with suspicion of primary focus in the follow-up period after PET/CT.
Metastatic lesions with increased tracer uptake comparable to surrounding normal tissues were deemed positive for metastatic spread. Conversely, nodular lesions with no detectable tracer uptake were deemed negative for metastatic spread, even if they are identified on the CT portion. This method of PET/CT image analysis was derived from the results of previous studies [17,18,19].
A diagnosis of the primary malignancy site was classified as true positive (TP) when it was confirmed histologically during the follow-up. If it was confirmed as benign or if the patient was without any signs of malignancy during the follow-up, the diagnosis was classified as false positive (FP). It was classified as true negative (TN) if neither FDG PET nor histological findings or clinical follow-up (including subsequent imaging tests) determined the site of the primary. When the site of the primary was not identified by FDG PET, but was proven histologically or by follow-up using other imaging studies, the finding was classified as being false negative (FN).
Histopathological evaluation
Biopsy/operation specimens were histopathologically evaluated by using standard histomorphometric techniques. All specimens were sliced, routinely processed, stained with hematoxylin eosin, examined microscopically, and interpreted by an experienced pathologist in oncologic pathology.
Statistical analysis
For the sample size, based on past review of literature, Kwee et al. showed the specificity of PET/CT to range from 73 to 100% [3] and the prevalence of carcinoma of unknown primary tumors (CUP) ranges from 0.5 to 9% of all patients with malignant neoplasms according to Le Chevalier et al. [20]. Sample size has been calculated at 80% power and 95%CI, and it is estimated that 180 patients would be required. Sample size (n) based on
$$ \mathrm{Specificity}=Z21-\alpha /2\times \mathrm{SP}\times \left(1-\mathrm{SP}\right)L2\times \left(1-\mathrm{Prevalence}\right) $$
where n = required sample size, S = anticipated specificity, α = size of the critical region (1 − α is the confidence level), z1 − α/2 = standard normal deviate corresponding to the specified size of the critical region (α), and L = absolute precision desired on either side (half-width of the confidence interval) of specificity. In this study, 5 patients were dropped out and were unable to do PET/CT examination: inability to cooperate with the scan process “inability to lie relatively still for 1–2 h and to lie supine for 30–60 min due to severe bone pain” (2 patients), allergy to intravenous contrast (2 patients), diabetic patients with uncontrolled blood glucose levels (1 patient).
Data were analyzed using IBM© SPSS© Statistics version 23 (IBM© Corp., Armonk, NY). Numerical and categorical data were presented as number or proportion and percentage. All values are presented as median and range. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy with 95% confidence intervals (CI) were calculated by using standard statistical formulas by standard 2 × 2 tables. The positive predictive value (PPV) of PET-CT-guided biopsy was calculated by determining the percentage of biopsies from primary sites detected by PET-CT scan that showed histopathological evidence of malignancy. A confidence level of less than 0.05 was considered to be statistically significant. The sensitivity, specificity, accuracy, and detection rate of PET/CT in detecting primary malignancy were calculated using the following statistical formulae. Sensitivity = TP/(TP + FN), specificity = TN/(TN + FP), and accuracy = (TP + TN)/(TP + FP + TN + FN) [21].