Establishment of Crystal Digital PCR Three-channel Detection Method for HER-2 Gene Copy Number Variation

Breast cancer is the number one common malignant tumor in women

Tri-color fluorescence channel Crystal microdrop chip digital PCR for detection of HER2 copy number amplification

The amplification of HER2 is a crucial indicator for targeted therapy in breast cancer.

Next, we will introduce how the Crystal droplet chip digital PCR can reliably identify the HER2 amplification in the small tumor lesion samples?

We optimized the previously reported Assay on the Crystal digital PCR platform to detect HER2 (ERBB2) and MRMM1 on chromosome 17, and TSN (internal reference gene) on chromosome 2 [1]

Sensitivity of HER2 amplification detection

Genomic DNA extracted from the SKBR3 cell line (HER2/TSN ratio 10:1) was incorporated into normal genomic DNA to achieve a mutation frequency ranging from 1% to 12%, and a model with a HER2/TSN ratio ranging from 1 to 2.

Use the Poisson formula to calculate the absolute concentration of TSN and HER2, and apply the Z-test to test the significance of the log HER2/TSN ratio (approximately normal distribution) characterizing the amount of mutant DNA (α=β=5%) [2]

Figure 1.

Detection of HER2 amplification and polyploidy of chromosome 17 in breast cancer patient samples

The current method of HER2 amplification in tumor samples relies on immunohistochemistry (IHC) to assess HER2 overexpression.

In this study, DNA extracted from 7 breast cancer patient samples provided by the French Gustave Roussy Institute was used for detection, and the results obtained by Crystal digital PCR were compared with those obtained by standard diagnostic procedures

Figure 2.

The results of IHC/FISH and Crystal digital PCR on patient samples 1-4 (both methods are considered positive for HER2 amplification) are consistent with samples from patients 6 and 7 (negative for HER2 amplification)

However, standard diagnostic procedures detected the amplification of HER2 in the sample of patient 5, and Crystal digital PCR detected an increase in the HER2 signal due to the polysomy of chromosome 17

Comparison with other digital PCR platforms

Using the independently developed Her2 gene detection Assay, by comparing the detection of 16 breast cancer patient samples with the IHC method and different digital PCR technologies, the Crystal digital PCR technology detects HER-2 with a sensitivity of 75% and a specificity of 92%.

At the same time, it can overcome the heterogeneity problem involved in the cytological method, the operation is simpler, and the cost is reduced by 75% compared with the traditional method

Figure 3.

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