Detection of clinical progression through plasma ctDNA in metastatic melanoma patients: A comparison to radiological progression

Background The validity of circulating tumour DNA (ctDNA) as an indicator of disease progression compared to medical imaging in patients with metastatic melanoma requires detailed evaluation. Methods Here, we carried out a retrospective ctDNA analysis of 108 plasma samples collected at the time of disease progression. We also analysed a validation cohort of 66 metastatic melanoma patients monitored prospectively after response to systemic therapy. Results ctDNA was detected in 62% of patients at the time of disease progression. For 67 patients that responded to treatment, the mean ctDNA level at progressive disease was significantly higher than at the time of response (P \u3c 0.0001). However, only 30 of these 67 (45%) patients had a statistically significant increase in ctDNA by Poisson test. A validation cohort of 66 metastatic melanoma patients monitored prospectively indicated a 56% detection rate of ctDNA at progression, with only two cases showing increased ctDNA prior to radiological progression. Finally, a correlation between ctDNA levels and metabolic tumour burden was only observed in treatment naïve patients but not at the time of progression in a subgroup of patients failing BRAF inhibition (N = 15). Conclusions These results highlight the low efficacy of ctDNA to detect disease progression in melanoma when compared mainly to standard positron emission tomography imaging

Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma

Background: Currently mainly BRAF mutant circulating tumor DNA (ctDNA) is utilized to monitor patients with melanoma. TERT promoter mutations are common in various cancers and found in up to 70% of melanomas, including half of BRAF wildtype cases. Therefore, a sensitive method for detection of TERT promoter mutations would increase the number of patients that could be monitored through ctDNA analysis. Methods: A droplet digital PCR (ddPCR) assay was designed for the concurrent detection of chr5:1,295,228 C \u3e T and chr5:1,295,250 C \u3e T TERT promoter mutations. The assay was validated using 39 melanoma cell lines and 22 matched plasma and tumor samples. In addition, plasma samples from 56 metastatic melanoma patients and 56 healthy controls were tested for TERT promoter mutations. Results: The established ddPCR assay detected TERT promoter mutations with a lower limit of detection (LOD) of 0.17%. Total concordance was demonstrated between ddPCR and Sanger sequencing in all cell lines except one, which carried a second mutation within the probe binding-site. Concordance between matched plasma and tumor tissue was 68% (15/22), with a sensitivity of 53% (95% CI, 27%- 79%) and a specificity of 100% (95% CI, 59%-100%). A significantly longer PFS (p=0.028) was evident in ctDNA negative patients. Importantly, our TERT promoter mutations ddPCR assay allowed detection of ctDNA in 11 BRAF wild-type cases. Conclusions: The TERT promoter mutation ddPCR assay offers a sensitive test for molecular analysis of melanoma tumors and ctDNA, with the potential to be applied to other cancers

The prognostic impact of circulating tumour dna in melanoma patients treated with systemic therapies—beyond braf mutant detection

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. In this study, we evaluated the predictive value of circulating tumour DNA (ctDNA) to inform therapeutic outcomes in metastatic melanoma patients receiving systemic therapies. We analysed 142 plasma samples from metastatic melanoma patients prior to commencement of systemic therapy: 70 were treated with BRAF/MEK inhibitors and 72 with immunotherapies. Patient-specific droplet digital polymerase chain reaction assays were designed for ctDNA detection. Plasma ctDNA was detected in 56% of patients prior to first-line anti-PD1 and/or anti-CTLA-4 treatment. The detection rate in the immunotherapy cohort was comparably lower than those with BRAF inhibitors (76%, p = 0.0149). Decreasing ctDNA levels within 12 weeks of treatment was strongly concordant with treatment response (Cohen’s k = 0.798, p \u3c 0.001) and predictive of longer progression free survival. Notably, a slower kinetic of ctDNA decline was observed in patients treated with immunotherapy compared to those on BRAF/MEK inhibitors. Whole exome sequencing of ctDNA was also conducted in 9 patients commencing anti-PD-1 therapy to derive tumour mutational burden (TMB) and neoepitope load measurements. The results showed a trend of high TMB and neoepitope load in responders compared to non-responders. Overall, our data suggest that changes in ctDNA can serve as an early indicator of outcomes in metastatic melanoma patients treated with systemic therapies and therefore may serve as a tool to guide treatment decisions

Circulating Melanoma Cell Subpopulations: Their Heterogeneity and Differential Responses to Treatment

Metastatic melanoma is a highly heterogeneous tumor; thus, methods to analyze tumor-derived cells circulating in blood should address this diversity. Taking this into account, we analyzed, using multiparametric flow cytometry, the co-expression of the melanoma markers melanoma cell adhesion molecule and melanoma-associated chondroitin sulphate proteoglycan and the tumor-initiating markers ATP-binding cassette sub-family B member 5 (ABCB5), CD271, and receptor activator of NF-κβ (RANK) in individual circulating tumor cells (CTCs) from 40 late-stage (III–IV) and 16 early-stage (I–II) melanoma patients. CTCs were heterogeneous within and between patients, with limited co-expression between the five markers analyzed. Analysis of patient matched blood and metastatic tumors revealed that ABCB5 and RANK subpopulations are more common among CTCs than in the solid tumors, suggesting a preferential selection for these cells in circulation. Pairwise comparison of CTC subpopulations longitudinally before and 6–13 weeks after treatment initiation showed that the percentage of RANK+ CTCs significantly increased in the patients undergoing targeted therapy (N=16, P<0.01). Moreover, the presence of ⩾5 RANK+ CTCs in the blood of patients undergoing targeted therapies was prognostic of shorter progression-free survival (hazards ratio 8.73, 95% confidence interval 1.82–41.75, P<0.01). Taken together, our results provide evidence of the heterogeneity among CTC subpopulations in melanoma and the differential response of these subpopulations to targeted therapy

Detection of metastases using circulating tumour DNA in uveal melanoma

Background: Approximately 50% of uveal melanoma (UM) patients will develop metastatic disease depending on the genetic features of the primary tumour. Patients need 3–12 monthly scans, depending on their prognosis, which is costly and often non-specific. Circulating tumour DNA (ctDNA) quantification could serve as a test to detect and monitor patients for early signs of metastasis and therapeutic response. Methods: We assessed ctDNA as a biomarker in three distinct UM cohorts using droplet-digital PCR: (A) a retrospective analysis of primary UM patients to predict metastases; (B) a prospective analysis of UM patients after resolution of their primary tumour for early detection of metastases; and (C) monitoring treatment response in metastatic UM patients. Results: Cohort A: ctDNA levels were not associated with the development of metastases. Cohort B: ctDNA was detected in 17/25 (68%) with radiological diagnosis of metastases. ctDNA was the strongest predictor of overall survival in a multivariate analysis (HR = 15.8, 95% CI 1.7–151.2, p = 0.017). Cohort C: ctDNA monitoring of patients undergoing immunotherapy revealed a reduction in the levels of ctDNA in patients with combination immunotherapy. Conclusions: Our proof-of-concept study shows the biomarker feasibility potential of ctDNA monitoring in for the clinical management of uveal melanoma patients

Intra- and intertumoral heterogeneity of liver metastases in a patient with uveal melanoma revealed by single-cell RNA sequencing

Tumor heterogeneity is a major obstacle to the success of cancer treatment. An accurate understanding and recognition of tumor heterogeneity is critical in the clinical management of cancer patients. Here, we utilized single-cell RNA sequencing (scRNA-seq) to uncover the intra- and intertumoral heterogeneity of liver metastases from a patient with metastatic uveal melanoma. The two metastases analyzed were largely infiltrated by noncancerous cells with significant variability in the proportion of different cell types. Analysis of copy-number variations (CNVs) showed gain of 8q and loss of 6q in both tumors, but loss of Chromosome 3 was only detected in one of the tumors. Single-nucleotide polymorphism (SNP) array revealed a uniparental isodisomy 3 in the tumor with two copies of Chromosome 3, indicating a regain of Chromosome 3 during the development of the metastatic disease. In addition, both tumors harbored subclones with additional CNVs. Pathway enrichment analysis of differentially expressed genes revealed that cancer cells in the metastasis with isodisomy 3 showed up-regulation in epithelial–mesenchymal transition and myogenesis related genes. In contrast, up-regulation in interferon signaling was observed in the metastasis with monosomy 3 and increased T-cell infiltrate. This study highlights the complexity and heterogeneity of different metastases within an individual case of uveal melanoma

Prognostic value of HLA-I homozygosity in patients with non-small cell lung cancer treated with single agent immunotherapy

Background: We aimed to assess the impact of genomic human leukocyte antigen (HLA)-I/II homozygosity on the survival benefit of patients with unresectable locally advanced, metastatic non-small lung cancer treated by single-agent programmed cell death protein-1/programmed death ligand 1 (PD1/PDL1) inhibitors. Methods: We collected blood from 170 patients with advanced lung cancer treated with immunotherapy at two major oncology centers in Western Australia. Genomic DNA was extracted from white blood cells and used for HLA-I/II high-resolution typing. HLA-I/II homozygosity was tested for association with survival outcomes. Univariable and multivariable Cox regression models were constructed to determine whether HLA homozygosity was an independent prognostic factor affecting Overall Survival (OS) and Progression Free Survival (PFS). We also investigated the association between individual HLA-A and -B supertypes with OS. Results: Homozygosity at HLA-I loci, but not HLA-II, was significantly associated with shorter OS (HR=2.17, 95% CI 1.13 to 4.17, p=0.02) in both univariable and multivariable analysis. The effect of HLA-I homozygosity in OS was particularly relevant for patients with tumors expressing PDL1 ≥50% (HR=3.93, 95% CI 1.30 to 11.85, p<0.001). The adverse effect of HLA-I homozygosity on PFS was only apparent after controlling for interactions between PDL1 status and HLA-I genotype (HR=2.21, 95% CI 1.04 to 4.70, p=0.038). The presence of HLA-A02 supertype was the only HLA-I supertype to be associated with improved OS (HR=0.56, 95% CI 0.34 to 0.93, p=0.023). Conclusion: Our results suggest that homozygosity at ≥1 HLA-I loci is associated with short OS and PFS in patients with advanced non-small cell lung cancer with PDL1 ≥50% treated with single-agent immunotherapy. Carriers of HLA-A02 supertype reported better survival outcomes in this cohort of patients

301MO Genomic HLA as a predictive biomarker for survival among non-small cell lung cancer patient treated with single agent immunotherapy

We aimed to assess the role of genomic HLA-I/II homozygosity in the overall survival benefit in patients with unresectable locally advanced, metastatic non-small lung cancer treated by single agent PD1/PDL1 inhibitors..