Clinical validation and implementation of droplet digital PCR for the detection of BRAF mutations from cell-free DNA.

Droplet digital PCR (ddPCR) has been demonstrated in many research studies to be a sensitive method in the analysis of circulating tumour DNA (ctDNA) for identifying mutations and tracking disease. The transition of ddPCR into the diagnostic setting requires a number of critical steps including the assessment of accuracy and precision and ultimately implementation into clinical use. Here we present the clinical validation of ddPCR for the detection of BRAF mutations (V600E and V600K) from plasma. We describe the performance characteristics assessed including the limit of blank, limit of detection, ruggedness, accuracy, precision and the effect of the matrix. Overall, each assay could achieve a limit of detection of 0.5% variant allele fraction and was highly accurate, with 100% concordance of results obtained from routine diagnostic testing of formalin fixed tumour samples or reference controls (n=36 for BRAF V600E and n=30 for BRAF V600K). Inter-laboratory reproducibility across 12 plasma samples for each assay was also assessed and results were 100% concordant. Overall, we report the successful validation and translation of a ddPCR assay into clinical routine practice.

Detection of cell-free microbial DNA using a contaminant-controlled analysis framework.

BACKGROUND: The human microbiome plays an important role in cancer. Accumulating evidence indicates that commensal microbiome-derived DNA may be represented in minute quantities in the cell-free DNA of human blood and could possibly be harnessed as a new cancer biomarker. However, there has been limited use of rigorous experimental controls to account for contamination, which invariably affects low-biomass microbiome studies. RESULTS: We apply a combination of 16S-rRNA-gene sequencing and droplet digital PCR to determine if the specific detection of cell-free microbial DNA (cfmDNA) is possible in metastatic melanoma patients. Compared to matched stool and saliva samples, the absolute concentration of cfmDNA is low but significantly above the levels detected from negative controls. The microbial community of plasma is strongly influenced by laboratory and reagent contaminants introduced during the DNA extraction and sequencing processes. Through the application of an in silico decontamination strategy including the filtering of amplicon sequence variants (ASVs) with batch dependent abundances and those with a higher prevalence in negative controls, we identify known gut commensal bacteria, such as Faecalibacterium, Bacteroides and Ruminococcus, and also other uncharacterised ASVs. We analyse additional plasma samples, highlighting the potential of this framework to identify differences in cfmDNA between healthy and cancer patients. CONCLUSIONS: Together, these observations indicate that plasma can harbour a low yet detectable level of cfmDNA. The results highlight the importance of accounting for contamination and provide an analytical decontamination framework to allow the accurate detection of cfmDNA for future biomarker studies in cancer and other diseases.

Evolution of late-stage metastatic melanoma is dominated by aneuploidy and whole genome doubling.

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.

γδ T Cells in Merkel Cell Carcinomas Have a Proinflammatory Profile Prognostic of Patient Survival.

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4+ or CD8+ T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2- γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.See related Spotlight on p. 600.

Clinical, FDG-PET and molecular markers of immune checkpoint inhibitor response in patients with metastatic Merkel cell carcinoma.

BACKGROUND: Metastatic Merkel cell carcinoma (mMCC) is an aggressive neuroendocrine malignancy of the skin with a poor prognosis. Immune checkpoint inhibitors (ICIs) have shown substantial efficacy and favorable safety in clinical trials. METHODS: Medical records of patients (pts) with mMCC treated with ICIs from August 2015 to December 2018 at Peter MacCallum Cancer Centre in Australia were analyzed. Response was assessed with serial imaging, the majority with FDG-PET/CT scans. RNA sequencing and immunohistochemistry for PD-L1, CD3 and Merkel cell polyomavirus (MCPyV) on tumor samples was performed. RESULTS: 23 pts with mMCC were treated with ICIs. A median of 8 cycles (range 1 to 47) were administered, with treatment ongoing in 6 pts. Objective responses (OR) were observed in 14 pts (61%): 10 (44%) complete responses (CR) and 4 (17%) partial responses (PR). Median time to response was 8 weeks (range 6 to 12) and 12-month progression-free survival rate was 39%. Increased OR were seen in pts aged less than 75 (OR 80% vs 46%), no prior history of chemotherapy (OR 64% vs 50%), patients with an immune-related adverse event (OR 100% vs 43%) and in MCPyV-negative tumors (OR 69% vs 43%). Pts with a CR had lower mean metabolic tumor volume on baseline FDG-PET/CT scan (CR: 35.7 mL, no CR: 187.8 mL, p=0.05). There was no correlation between PD-L1 positivity and MCPyV status (p=0.764) or OR (p=0.245). 10 pts received radiation therapy (RT) during ICI: 4 pts started RT concurrently (OR 75%, CR 50%), 3 pts had isolated ICI-resistant lesions successfully treated with RT and 3 pts with multisite progression continued to progress despite RT. Overall, 6 pts (26%) had grade 1-2 immune-related adverse events. CONCLUSION: ICIs showed efficacy and safety in mMCC consistent with trial data. Clinical and imaging predictors of response were identified.

The survivorship experience for patients with metastatic melanoma on immune checkpoint and BRAF-MEK inhibitors.

PURPOSE: Immune checkpoint inhibitors (ICI) and BRAF and MEK inhibitors (BMi) have improved survival in metastatic melanoma (MM). However, the experience of long-term responders remains undescribed. This study characterised survivorship issues faced by long-term responders to ICI or BMi. METHODS: Patients with MM, aged ≥ 18 years old, ≥ 6 months post-ICI or BMi initiation with an objective response or stable disease. A 72-question survey assessed physical and psychological effects, impact on lifestyle, access to information, satisfaction with care, and availability of supports. RESULTS: One hundred and five of 120 (88%) patients completed the survey (ICI 69/BMI 36). For the ICI cohort, 39 (57%) were receiving ongoing treatment, 17 ceased due to toxicity and 13 due to a sustained response. For the BMi cohort, 31 (85%) were receiving ongoing treatment, 4 ceased due to toxicity and 1 due to a sustained complete response. At data cut-off on 18 December 2018, median PFS (range) was 2.5 years (1.3-8.5) for ICI and 3.1 years (0.6-7.3) for BMi. Long-term toxicities included dry/itchy skin (ICI 51, 74%/ BMi 25, 69%), arthralgias (ICI 30, 58%/ BMi 23, 64%) and fatigue (ICI 62, 90%/ BMi 33, 92%). Psychological morbidity was common, including anxiety awaiting results (ICI 50, 72%/ BMi 29, 81%), fear of melanoma recurring or progressing (ICI 56, 81%/ BMi 31, 86%) or death (ICI 44, 64%/ BMi 26, 72%). CONCLUSION: MM survivors experience chronic treatment toxicities and frequently report psychological concerns. IMPLICATIONS FOR CANCER SURVIVORS: Survivors may benefit from discussions regarding long-term toxicities and tailored psychological supports.

Genomic Analysis of Circulating Tumor DNA Using a Melanoma-Specific UltraSEEK Oncogene Panel.

The analysis of circulating tumor DNA provides a minimally invasive molecular interrogation that has the potential to guide treatment selection and disease monitoring. Here, the authors evaluated a custom UltraSEEK melanoma panel for the MassARRAY system, probing for 61 mutations over 13 genes. The analytical sensitivity and clinical accuracy of the UltraSEEK melanoma panel was compared with droplet digital PCR. The blinded analysis of 68 mutations detected in 48 plasma samples from stage IV melanoma patients revealed a concordance of 88% between the two platforms. Further comparison of both methods for the detection of BRAF V600E mutations in 77 plasma samples demonstrated a Cohen's κ of 0.826 (bias-corrected and accelerated 95% CI, 0.669-0.946). These results indicate that the UltraSEEK melanoma panel is as sensitive as droplet digital PCR for the detection of circulating tumor DNA in this cohort of patients but highlight the need for detected variants to be confirmed orthogonally to mitigate any false-positive results. The MassARRAY system enables rapid and sensitive genotyping for the detection of multiple melanoma-associated mutations in plasma.

Circulating Tumor DNA Analysis and Functional Imaging Provide Complementary Approaches for Comprehensive Disease Monitoring in Metastatic Melanoma.

PURPOSE: Circulating tumor DNA (ctDNA) allows noninvasive disease monitoring across a range of malignancies. In metastatic melanoma, the extent to which ctDNA reflects changes in metabolic disease burden assessed by 18F-labeled fluorodeoxyglucose positron emission tomography (FDG-PET) is unknown. We assessed the role of ctDNA analysis in combination with FDG-PET to monitor tumor burden and genomic heterogeneity throughout treatment. PATIENTS AND METHODS: We performed a comprehensive analysis of serial ctDNA and FDG-PET in 52 patients who received systemic therapy for metastatic melanoma. Next-generation sequencing and digital polymerase chain reaction were used to analyze plasma samples from the cohort. RESULTS: ctDNA levels were monitored across patients with mutant BRAF, NRAS, and BRAF/NRAS wild type disease. Mutant BRAF and NRAS ctDNA levels correlated closely with changes in metabolic disease burden throughout treatment. TERT promoter mutant ctDNA levels also paralleled changes in tumor burden, which provide an alternative marker for disease monitoring. Of note, subcutaneous and cerebral disease sites were not well represented in plasma. Early changes in ctDNA and metabolic disease burden were important indicators of treatment response. Patients with an early decrease in ctDNA post-treatment had improved progression-free survival compared with patients in whom ctDNA levels remained unchanged or increased over time (hazard ratio, 2.6; P = .05). ctDNA analysis contributed key molecular information through the identification of putative resistance mechanisms to targeted therapy. A detailed comparison of the genomic architecture of plasma and multiregional tumor biopsy specimens at autopsy revealed the ability of ctDNA to comprehensively capture genomic heterogeneity across multiple disease sites. CONCLUSION: The findings highlight the powerful role of ctDNA in metastatic melanoma as a complementary modality to functional imaging that allows real-time monitoring of both tumor burden and genomic changes throughout therapy.

Activity of trametinib in K601E and L597Q BRAF mutation-positive metastatic melanoma.

BRAF and MEK inhibitors are not established treatments for non-V600 mutation-positive metastatic melanoma. We carried out a retrospective analysis of efficacy and safety in four patients with BRAF K601E and one patient with L597Q mutation-positive metastatic melanoma treated with the MEK inhibitor trametinib. Three patients achieved a RECIST partial response, including the patient with an L597Q mutation. Paired biopsies available in one of the five patients showed reduced phospho-ERK signalling and this corresponded to a metabolic response on F-fluorodeoxyglucose-PET scanning. Trametinib toxicity was manageable. Trametinib has antitumour activity in patients with BRAF K601E and L597Q mutation-positive metastatic melanoma.

Sustained clinical responses to tyrosine kinase inhibitor sunitinib in thyroid carcinoma.

The limited therapeutic options available for patients with metastatic papillary thyroid carcinomas (PTC) and follicular thyroid carcinomas (FTC) necessitates the development of novel therapies. Identification of somatic rearrangements of the tyrosine kinase domain of the RET gene in PTC have improved our understanding of thyroid tumorigenesis. Sunitinib is active against the RET kinase and has both antineoplastic and antiangiogenic properties. Its role in the treatment of patients with thyroid carcinoma has yet to be evaluated in clinical trials. Two patients with progressive metastatic thyroid carcinoma (case 1: PTC, and case 2: FTC) were enroled in a phase I clinical trial to evaluate positron emission tomography (PET) in the monitoring of response to sunitinib. Tumour biopsies and PET were performed at baseline and 4 weeks after the commencement of sunitinib. Activation of the RET kinase pathway was evaluated using immunohistochemistry (IHC) and western blot analysis of total phosphorylated tyrosine and downstream signalling targets of the RET pathway. Both patients demonstrated sustained clinical responses to sunitinib over a duration of 4 years. In case 1, (PTC) PET confirmed evidence of a partial metabolic response, and IHC and western blot analysis demonstrated inhibition of the RET kinase pathway posttreatment. In case 2, (FTC) PET confirmed stable disease after sunitinib. IHC staining of the tumour showed low total phosphorylated tyrosine staining at baseline which did not change after treatment. These case studies highlight potential activity of sunitinib in patients with metastatic thyroid carcinoma. Sunitinib seems to be a promising agent in the treatment of thyroid cancers and this requires validation in future clinical trials.