CK19 Combined With Contrast-enhanced Ultrasound: a Prediction System on Axillary Lymph Node Metastasis in Breast Cancer
Conditions: Lymph Node, Breastcancer
Sponsor: Zhejiang Cancer Hospital

ID: ZJCH-CK19A 
    
 
Initial version (1.0) date: Nov.11, 2016 
Final version (2.0) date: Feb.2, 2017 
NCT02992067 
CK19 Combined With Contrast-enhanced Ultrasound: a 
Prediction System on Axillary Lymph Node Metastasis in 
Breast Cancer 
 
 
Study Protocol with SAP  
 
 
 

OBJECTIVE:  
Based on our previous research, we developed a model predicting the risk of 
lymph node involvement in early breast cancer patients by combining CK19 
mRNA in peripheral blood and Contrast-enhanced Ultrasound. 
DESIGN:  
Recruiting patients diagnosed cI, cII or cT3N1M0 (operable) invasive breast 
cancer as training set. In these patients, we tested the CK19 mRNA in peripheral 
blood and contrast-enhanced ultrasound (CEUS). With these two parameters and 
other clinical pathological characteristics, we can build a model calculating the 
risk of lymph node involvement by Logistic method. The predicting accurate, 
false negative rate will be evaluated firstly in the training set.  
METHODS: 
We identified cases diagnosed as operable invasive breast cancer from Dec 2016 
to Aug 2018 in Zhejiang Cancer Hospital. 
l Sampling Method: Probability Sample  
Minimum Age: 18 Years 
Maximum Age: 70 Years 
Sex:Female  
Gender Based: 
Accepts Healthy Volunteers: No  
l Criteria: 
Inclusion Criteria: 
female, age 18~70 years old 
the test group should be in accordance with the pathological diagnosis of breast 
cancer, the clinical stage of breast cancer (stage cTis, cI, cII and cT3N1M0), the 
initial treatment to receive surgical treatment. Complete preoperative clinical 
diagnosis data, including the ipsilateral axillary lymph node CEUS data and 
peripheral blood sample; The negative-control group should comply with the 
pathological diagnosis, breast adenosis, fibrocystic lesions, breast cysts and 
other benign lesions, with complete preoperative clinical data, also including 
ipsilateral axillary lymph node CEUS data and peripheral blood sample 
Exclusion criteria: 
metastatic breast cancer, inflammatory breast disease, surgery without lymph 
node staging 
pregnancy or lactation 

patients with hematopoietic system disease or cancer, autoimmune diseases 
preservation of substandard peripheral blood samples.  
l CK19 mRNA testing 
Peripheral blood sample collection  
Patient peripheral blood samples were collected one day before operation. A total of 
10 ml anticoagulated peripheral blood was taken from each patient. The first 2 ml of 
blood was discarded to avoid potential contamination by normal epithelial cells, and 
the remaining 8 ml was collected into 20-ml sterilized tubes containing EDTA.  
Specimen processing  
One ml of saline and 2 ml of lymphocyte separation medium were added to 1 ml of 
anticoagulated blood. Samples were centrifuged at 2500 rpm for 20 min after  
standing, and the white cell layer was drawn out and centrifuged at 12000 rpm for 5 
min. Trizol (1 ml) was added to the precipitate, and was fully mixed. We then added 
0.2 ml chloroform to the precipitate, which was mixed on a shaker at 4°C for 5 min 
and then centrifuged at 12000 rpm for 10 min. We collected 500 ul of the upper 
aqueous phase into a sterilized centrifuge tube and added an equal volume of 
isopropanol. The tube was gently mixed at 4°C for 10 min and then centrifuged at 
12000 rpm for 15 min.  
RNA preparation  
The RNA precipitate was washed with 1 ml of 75% ethanol and centrifuged at 7500 
rpm for 5 min at 4°C. Total cellular RNA was extracted by the acid guanidium 
thiocyanate-phenol-chloroform extraction procedure [20]. The RNA was resuspended 
in 25 ul of diethy pyrocarbonate (DEPC)-treated water and spectrophotometrically 
quantitated at 260 nm and 280 nm to assess purity. Extracted RNA was treated with 
RNase- free Dnase I (TaKaRa Biotechnology, Da Lian, China) according to the 
manufacturer’s instructions to remove contaminating DNA.  
Reverse transcription reaction  
CK19 reverse transcription was performed according to manufacture’s instructions. 
Briefly, 1 ug of RNA was reverse-transcribed for 15 min at 37°C in a 25 ul reaction 
mix containing 0.5 ul PrimeScript RT enzyme mix I, 0.5 ul oligo dT primer, 2 ul 
random 6-mers and 10 ul RNase free dH2O. The reaction  
was terminated by heating at 85°C for 5 sec.  
RT-PCR  

CK19 primers were designed from previously published sequences [21]. The sense 
primer, 5'-AAGCTA ACCATGCAGAACCTCAACGACCGC-3', and antisense 
primer, 5'-TTATTGGCAGGTCAGGAGAAGAGCC-3', were used for the first round 
of amplification. The second PCR amplification was carried out using the CK19 sense 
primer, 5'-TCCCGCGACTACAGCCACTACTAC ACGACC-3', and antisense 
primer, 5'-CGCGACTTGATG TCCATGAGCCGCTGGTAC-3'. PCR reaction 
conditions were as follows: 94°C for 3 min and 94°C for 30 sec for 40 cycles, 
followed by a final extension at 60°C for 35 sec. All RT-PCR products were 
separated by electrophoresis in a 1.5% agarose gel and were analyzed automatically 
via computer. CK19 levels were considered positive when copies ≥ 1000.  
l CEUS  
GE Logic E9 ultrasonic diagnostic instrument was adopted. During angiography 
with 9L probe, the transmitting frequency of the probe was 8.4-9.0mhz, and the 
mechanical index was 0.11. Parameters: 2 harmonics, gain 125, and time gain 
compensation remained unchanged during each angiography. SonoVue 
lyophilized powder (containing 59mg sulfur hexafluoride microbubbles, Bracco, 
Italy) was used for contrast. During the angiography, 1 SonoVue was taken, 5ml 
of normal saline was used to prepare the suspension, and the suspension was 
oscillated for 30s for later use. According to the results of general ultrasound 
examination, the axillary lymph nodes were scanned and the target suspected 
lesions were selected for display. After shaking the standby suspension well, 
2.4ml was extracted and injected through the anterior cubital vein group, and 
then 5ml normal saline was injected to flush the tube, and the movement and 
distribution of contrast agent microbubbles in the lesions were immediately 
observed. The dynamic observation time was 3 minutes, and the image was 
saved for analysis by ITC software. The results of each report were 
independently determined and recorded by two tumor ultrasound doctors with 
secondary or higher titles. According to the references and the European 
federation of ultrasound medicine and biology, ceus guidelines for non-liver 
clinical application, the following criteria were used to evaluate the scores :(1) 
lymph node morphology, short diameter >1cm or long/short diameter <2(1 
point), otherwise 0 point; (2) lymph node morphology, short diameter >1cm or 
long/short diameter <2(1 point); (2) cortex ≥3mm 1, cortex < 3mm 0; (3) mixed, 
non-uniform enhancement or no enhancement 2 points, 0 points of uniform 
enhancement; Calculate the total score and record it. Doctors in the two groups 
reported consistency test, random sampling of 30 cases (sampling rate of 10%), 
only one item in the three aspects of morphology, cortex and enhancement was 
inconsistent in the two cases, scoring coincidence rate reached 93.33%.  
STATISTICAL ANALYSIS PLAN (SAP) 
SPSS (v22.ibm ® Statistics) was used for data collection and statistical analysis. 
The general data hypothesis test level was set as a =0.05 (double-tailed), and H1 

hypothesis was accepted when P < 0.05, i.e. the statistical difference between the 
two samples was statistically significant. Calculation of sample size required for 
modeling (diagnostic experiment) : n= (U alpha/delta) 2× (1-p) ×P. U alpha: 
alpha =0.05, U alpha =Z alpha /2=1.960. In the modeling standard study, delta 
=0.06, P sensitivity =0.9, P specificity =0.8, plus 10% of the additional sample 
size, the result is 310.06. In SLNS and detected nSLN pathological diagnosis of 
lymph nodes (gold standard: positive/negative) as dependent variable, 
incorporated into the general clinical data, CK19 were a grade and CEUS, 
application of binary linear regression model, the input method (Enter) to screen 
effective modeling variables, Forward the method to estimate the maximum 
likelihood ratio (Forward LR, higher reliability, the parameter estimation and 
Wald statistics) to establish the forecast model of combined method of formula 
of calculating the SLNS and nSLN detected probability of each case goal. With the 
pathological diagnosis of lymph nodes as the gold standard, the four grid table 
method of diagnostic test was used to calculate the prediction effect indexes of 
the prediction model formula: sensitivity (SEN), specificity (SPE), accuracy (AR) 
and Youden index. The prediction efficiency test of the prediction model formula 
is: area under the ROC curve (AUC); Hosmer and Lemeshow test was used to test 
the goodness of fit of the prediction model formula.