Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital PCR for BRAF and NRAS mutations

Abstract

We assessed the utility of droplet digital PCR (ddPCR) to evaluate the potential of using circulating tumour DNA (ctDNA) as a post therapy monitoring tool in melanoma by comparing it to serum LDH levels and RECIST scores. ddPCR was shown to be reliable in distinguishing mutant from wild type alleles with no false positives. Subsequently, we quantified ctDNA ((V600E)BRAF,(V600K)BRAF or (Q61H)NRAS) in 6 stage IV melanoma patients across several time points during their treatment course. All tested patients had detectable ctDNA, which exhibited dynamic changes corresponding to the changes in their disease status. The ctDNA levels fell upon treatment response and rose with detectable disease progression. In our group of patients, ctDNA was more consistent and informative than LDH as a blood-based biomarker. In addition, BRAF mutant ctDNA as detected by ddPCR could be used diagnostically where the tumour block was unavailable. In conclusion, this study demonstrates the applicability of using ddPCR to detect and quantify ctDNA in the plasma of melanoma patients.

Figure 1. False positive determination for ^V600EBRAF, ^V600KBRAF and ^Q61HNRAS.

Graphs represent cumulative data for 8 repeats. Using known quantities of wild type (WT) DNA (nil, 10 ng and 100 ng of DNA) against both mutant and WT probes for (a) ^V600EBRAF, (b) ^V600KBRAF and (c) ^Q61HNRAS mutations. There was no detectable mutant droplet count and the amount of droplets corresponding to WT DNA rose in proportion to the amount of DNA used. (NTC: no-template control. Total: total droplets generated.)

Figure 2. ctDNA and LDH level monitoring with clinical follow up for patient (a) 1, (b) 2, (c) 3 and (d) 4.

ctDNA and LDH levels recorded with corresponding scan results. ctDNA level showed dynamic changes corresponding with disease progression. (Numbers in brackets correspond to the RECIST score. PR: partial response. PD: progressive disease. SD: stable disease.) The upper limit of normal LDH level in an adult is 250 IU/L. (a) Patient 1 showed a 98.3% decrease in ctDNA level following initiation of dabrafenib. The level stayed low as patient improved clinically with tumour shrinkage seen on PET scans with associated falling in RECIST score. (b) Patient 2 developed drug resistance evident with increasing tumour size. The ctDNA level also increased accordingly. However, patient’s LDH level fell paradoxically with rising tumour burden. (c) Patient 3 had multiple spinal metastases and we were unable to perform radiological scans to monitor the disease progression secondary to the metallic implant to stabilize the spine. The patient deteriorated despite new drug treatment (MK-3475). The LDH level fell initially after initiation of the new drug and then rose. The ctDNA level on the contrary never decreased with the new treatment. (d) Patient 4 started MK3475 after failing dabrafenib and trametinib with increasing intra and extra-cranial metastasis. All extracranial lesions responded well but the intracranial lesions continued to progress. This is reflected in the generally increasing ctDNA level, potentially representing remaining disease activity in treatment resistant intracranial lesions. The level fell when ipilimumab was added to the treatment regimen.

Figure 3. ctDNA and LDH level monitoring for patient 5 with corresponding radiological findings.

(a) Patient 5’s ctDNA level fell following treatment (CheckMate 067 trial; anti-PD-1 antibody nivolumab alone versus anti-CTLA-4 antibody ipilimumab alone versus combination of the two). The CT scans also showed disease regression with (b) red arrow points to lung metastasis on day 1 of treatment and (c) the corresponding site on day 263 with no evidence of disease. PET scan performed on day 188 showed no metabolically active lesions (Fig. 3a).

Figure 4. ctDNA and LDH level monitoring for patient 6 with corresponding radiological findings.

(a) Patient 6’s ctDNA level fell rapidly after initiation of ipilimumab consistent with tumour shrinkage. The LDH level started to fall one month later than ctDNA level. (b) The CT image shows multiple large liver metastases (red arrow). The RECIST score was 237 on day 1 of treatment. (c) On day 158 since initiation of ipilimumab, the liver metastases have shrunken significantly (RECIST score 110).

Figure 5. ddPCR results for patient 6.

Following initiation of ipilimumab, patient 6 had significantly lower amounts of ^Q61HNRAS in plasma. (Blue dots are ^Q61HNRAS, green dots are wild type NRAS, and grey dots are droplets without DNA of interest.)