Comparison of In-House Methods and Cobas BRAF V600 Mutation Assay in Melanoma Tumor Samples

BRAF V600 mutation status was determined by INCa molecular laboratories "in-house" methods and Cobas 4800 BRAF V600 mutation test. Samples were analysed as V600 mutation, No V600 mutation and Non evaluable. Additionally, the type of V600 mutation (E, K, R, D, E2, other V600 mutation, not specified) was also evaluated only by INCa molecular laboratory "in-house" method.

The type of tumor sample used for evaluation of BRAF V600 mutation whether it was a biopsy or surgical specimen were reported

The source of tumor sample for BRAF V600 mutation detection whether taken from internal or external pathology laboratory were reported

The external or internal pathology laboratories involved in the process of fixation or embedding of the tumor sample was evaluated.

Time taken from the sampling to the fixation of the tumor sample was reported in range of 0-2 hours, 2-6 hours, >6 hours and unknown. Number of samples falling in each of the class were reported.

The different types of fixative Excell, formol, alcohol formol acetic acid and other, used to fix the tumor samples were reported.

Fixation duration is defined as the amount of time required in hours for the fixation of a samples. The fixation duration was categorized as <6 hours, 6-24 hours and >24 hours and unknown. Number of samples falling in each category were reported

Slice thickness of all the tumour samples was measured. The slice thickness was measured in micrometer.

Dewaxing is a method to recover the DNA from samples. Dewaxing information was collected as "Yes, No or Missing"

The percentage of necrosis defined as the death of one or more cells in the analysed zone was reported.

The percentage of tumor cells in the given tumor sample were reported.

The tumor samples with presence of melanin were categorized as Important, Few, Medium and Absent.

This method assessed DNA from the tumor samples was extracted by Automated method or Manual method.

Median DNA elution volume microliters [mcl] was reported.

The DNA concentration in the tissue elute was measured in nanogram per microliter (ng/mcL).

The total DNA concentration extracted from the tissue was measured in nanogram (ng).

The method described the size of amplicon used. It was measured in base pairs (bp).

Allele-specific PCR, High Resolution Melting (HRM) + Sanger sequencing, Pyrosequencing, Sanger sequencing, Real time PCR, SNaPshot were used for BRAF V600 mutation detection.

Total number of samples for whom punch was used in 'in-house analytical' method are reported.

The mean of number of slices per sample when no punch was used are reported.

This In-house analytical method measured the time between receipt of samples to the result determination. It measured the time in days.

The working time required by the technician from the time of DNA extraction to the time to obtain the results was measured in hours.

The DNA concentration as assessed by COBAS 4800 BRAF V600 Mutation assay was reported. The unit used to measure the DNA concentration was nanogram/microlitre (ng/mcl)

The punch done during Cobas 4800 BRAF V600 Mutation Test on the sample was described as Yes or No.

This describes the Cobas 4800 BRAF V600 Mutation Test, for the mean of number of slices when No punch method, was used. Of the 420 samples, punch was Yes, for 45 samples and punch was No, for 375 samples.

This analytical method for cobas 4800 BRAF V600 Mutation Test measured the time between receipt of samples to the result determination. It measured the time in days.

This cobas 4800 BRAF V600 Mutation Test analytical method measures the working time required by the technician from the time of DNA extraction to the time to obtain the results. The time duration was measured in hours.

Crossing DNA, DNA from In-House method analysed with cobas, SNaPshot, DNA from cobas analysed with In-House method, external site control test, Sanger sequencing, Kit CE-IVD Therascreen RGQ Qiagen, Kit Therascreen RGQ BRAF + Pyrosequencing by another platform (PF), Pyrosequencing, Mutation detection On Another Block, (primitive tumor [prm. tmr]), Sequencing And Therascreen kit (Qiagen) were used for management of discordance between "in-house" method and Cobas 4800 mutation test.

The final results obtained by discordance management of the 28 discordant samples were BRAF V600 mutation, No BRAF V600 mutation and Non-evaluable. These results were further assessed by the Investigator and interpreted as final result.