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.

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis. n = number of samples with BRAF V600 mutation by in-house method.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 Months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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.

Time frame: Up to 6 months

Population: The discordant sample population is defined as the samples whose result for BRAF V600 mutation by the in-house method did not show similar outcome with the cobas 4800 mutation test.

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.

Time frame: Up to 6 months

Population: The discordant sample population is defined as the samples whose result for BRAF V600 mutation by the in-house method did not show similar outcome with the cobas 4800 mutation test.

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)

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis. Data for the samples where the punch was not used were considered for analysis.

Median DNA elution volume microliters \[mcl\] was reported.

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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.

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis. Only 341 samples out of 420 were analysed as the information on presence of necrosis was missing for 79 samples in the assessed zones.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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.

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis. Data for the samples where the punch was No=375, was used for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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.

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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.

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis. Only available samples were included for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.

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

Time frame: Up to 6 months

Population: All samples meeting the inclusion criteria and not meeting the exclusion criteria were considered for analysis.