Advances in therapeutic approaches for melanoma urge the need for biomarkers that can identify patients at risk for recurrence and to guide treatment. The potential use of liquid biopsies in ...identifying biomarkers is increasingly being recognized. Here, we present a head-to-head comparison of several techniques to analyze circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in 20 patients with metastatic melanoma. In this study, we investigated whether diagnostic leukapheresis (DLA) combined with multimarker flow cytometry (FCM) increased the detection of CTCs in blood compared to the CellSearch platform. Additionally, we characterized cfDNA at the level of somatic mutations, extent of aneuploidy and genome-wide DNA methylation. Both CTCs and cfDNA measures were compared to tumor markers and extracranial tumor burden on radiological imaging. Compared to the CellSearch method applied on peripheral blood, DLA combined with FCM increased the proportion of patients with detectable CTCs from 35% to 70% (P = 0.06). However, the median percentage of cells that could be recovered by the DLA procedure was 29%. Alternatively, cfDNA mutation and methylation analysis detected tumor load in the majority of patients (90% and 93% of samples successfully analyzed, respectively). The aneuploidy score was positive in 35% of all patients. From all tumor measurements in blood, lactate dehydrogenase (LDH) levels were significantly correlated to variant allele frequency (P = 0.004). Furthermore, the presence of CTCs in DLA was associated with tumor burden (P < 0.001), whereas the presence of CTCs in peripheral blood was associated with number of lesions on radiological imaging (P < 0.001). In conclusion, DLA tended to increase the proportion of patients with detectable CTCs but was also associated with low recovery. Both cfDNA and CTCs were correlated with clinical parameters such as LDH levels and extracranial tumor burden.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Cervical screening by high-risk HPV (hrHPV) testing requires additional risk stratification (triage), as most infections are transient and only a subset of hrHPV-positive women harbours clinically ...relevant disease. Molecular triage markers such as microRNAs (miRNAs) and DNA methylation markers are particularly promising, as they can be objectively tested directly on hrHPV-positive scrapes and cervicovaginal self-samples. Here, we evaluated the marker potential of 10 candidate miRNAs in 209 hrHPV-positive scrapes of women with underlying precancer (cervical intraepithelial neoplasia, grade 2-3 (CIN2-3)), cancer, or without disease (CIN0/1). A predictive miRNA classifier for CIN3 detection was built using logistic regression, which was compared to and combined with DNA methylation marker FAM19A4. Markers were correlated to histology parameters and hrHPV genotype. A miRNA classifier consisting of miR-149, miR-20a, and miR-93 achieved an area under the curve (AUC) of 0.834 for CIN3 detection, which was not significantly different to that of FAM19A4 methylation (AUC: 0.862, p = 0.591). Combining miRNA and methylation analysis demonstrated complementarity between both marker types (AUC: 0.939). While the miRNA classifier seemed more predictive for CIN2, FAM19A4 methylation was particularly high in HPV16-positive and histologically advanced CIN3, i.e. CIN3 with high lesion volume. The miRNA classifier, FAM19A4 methylation, and the miRNA/methylation combination were highest in cancer-associated scrapes. In conclusion, a panel of three miRNAs is discriminatory for CIN3 in hrHPV-positive scrapes and can complement DNA methylation analysis for the efficient detection of cervical disease. Combined analysis of the two marker types warrants further evaluation as triage strategy in hrHPV-based screening.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
miRNAs represent an emerging class of promising biomarkers for cancer diagnostics. To perform reliable miRNA expression analysis using quantitative PCR, adequate data normalization is essential to ...remove nonbiological, technical variations. Ideal reference genes should be biologically stable and reduce technical variability of miRNA expression analysis. Herein is a new strategy for the identification and evaluation of reference genes that can be applied for miRNA-based diagnostic tests without entailing excessive additional experiments. We analyzed the expression of 11 carefully selected candidate reference genes in different types of cervical specimens ie, tissues, scrapes, and self-collected cervicovaginal specimens (self-samples). To identify the biologically most stable reference genes, three commonly used algorithms (GeNorm, NormFinder, and BestKeeper) were combined. Signal-to-noise ratios and P values between control and disease groups were calculated to validate the reduction in technical variability on expression analysis of two marker miRNAs. miR-423 was identified as a suitable reference gene for all sample types, to be used in combination with RNU24 in cervical tissues, RNU43 in scrapes, and miR-30b in self-samples. These findings demonstrate that the choice of reference genes may differ between different types of specimens, even when originating from the same anatomical source. More important, it is shown that adequate normalization increases the signal-to-noise ratio, which is not observed when normalizing to commonly used reference genes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
This prospective cohort study reports aneuploidy score by mFast-SeqS as a strong prognostic marker in MBC patients. mFAST-SeqS is an affordable and easily implementable method for the assessment of ...total ctDNA levels and, as such, provides an alternative prognostic tool. One mixed cohort (cohort A, n = 45) starting any type of treatment in any line of therapy and one larger cohort (cohort B, n = 129) consisting of patients starting aromatase inhibitors (AI) as first-line therapy were used. mFAST-SeqS was performed using plasma of blood in which CTCs (CellSearch) were enumerated. The resulting aneuploidy score was correlated with categorized CTC count and associated with outcome. The aneuploidy score was significantly correlated with CTC count, but discordance was observed in 31.6% when applying cut-offs of 5. In both cohorts, aneuploidy score was a significant prognostic marker for both PFS and OS. In the Cox regression models, the HR for aneuploidy score for PFS was 2.52 (95% CI: 1.56-4.07), and the HR for OS was 2.37 (95% CI: 1.36-4.14). Results presented here warrant further investigations into the clinical utility of this marker in MBC patients.
Given the lower specificity for high‐grade cervical lesions of high‐risk human papillomavirus (hrHPV) testing compared to cytology, additional triage testing for hrHPV test‐positive women is needed ...to detect high‐grade cervical lesions. Here, we tested whether combined methylation analysis for cell adhesion molecule 1 (CADM1) and T‐lymphocyte maturation associated protein (MAL), both functionally involved in cervical carcinogenesis, could serve as such a triage marker. Four quantitative methylation‐specific PCRs (qMSP), two for CADM1 (regions M12 and M18) and MAL (regions M1 and M2) each, were applied to 261 cervical tissue specimens ranging from no neoplasia to carcinoma. When qMSPs were combined and positivity for at least one of the qMSPs in the combination was taken into account, the highest positivity rates for cervical intraepithelial neoplasia grade 3 (CIN3) lesions (97%) and squamous cell‐ and adeno‐carcinomas (99%) were obtained by combining a single CADM1 marker with a single MAL marker. Subsequent qMSP analysis of 70 GP5+/6+‐PCR hrHPV‐positive scrapings revealed that a two‐marker panel consisting of CADM1‐M18 and MAL‐M1 was most discriminative, detecting 90% of women with CIN3 (n = 30), whereas it showed a positive result in only 13.5% of women without cervical disease (n = 40). Finally, we applied hrHPV GP5+/6+‐PCR testing followed by CADM1‐M18/MAL‐M1 methylation analysis to a cohort of 79 women visiting the outpatient colposcopy clinic. hrHPV testing revealed a sensitivity of 97% and a specificity of 33% for CIN3+. Additional CADM1‐M18/MAL‐M1 methylation analysis on the hrHPV‐positive women increased the specificity to 78% with a sensitivity of 70%. In conclusion, the methylation marker panel CADM1‐M18 and MAL‐M1 may serve as an alternative molecular triage tool for hrHPV‐positive women.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The addition of two years of abemaciclib treatment to standard adjuvant endocrine therapy in all patients with high risk ER+, HER2- early breast cancer (EBC) has been approved by the US Food and Drug ...Administration (FDA). Pre-selection of patients with an immediate risk of recurrence within the group of clinically high risk patients using detection of minimal residual disease (MRD) using patient-informed circulating tumor DNA assays during follow-up could enhance efficacy. Here, we investigate the cost-effectiveness of the addition of two years abemaciclib in all high risk HR+, HER2- patients and in MRD-guided high risk patients only.
Two semi-Markov models were developed to evaluate the cost-effectiveness of adding two years of abemaciclib compared to "standard treatment": 1) "abemaciclib all" and 2) "MRD-guided abemaciclib" using MRD-guidance. Data of the MonarchE trial were used to model the invasive disease-free survival (iDFS). Since iDFS and overall survival (OS) data of abemaciclib were currently limited, abemaciclib effects were extrapolated using a favorable, intermediate and unfavorable effect scenario.
The addition of abemaciclib in all high-risk EBC patients prolonged iDFS slightly (0.04 additional quality adjusted life years (QALYs)) and led to higher costs compared to standard ET, leading to a high incremental cost effectiveness ratio (ICER) of €1,551,876/QALY. Neither the favorable effect scenario (additional 1.09 QALYs) was cost-effective (ICER €62,935/QALY), using a willingness-to-pay threshold of €50,000/QALY. The "MRD-guided abemaciclib" strategy resulted in lower costs and an increase in QALYs (1.27) compared to "standard treatment" in the unfavorable effect scenario.
The addition of abemaciclib to adjuvant endocrine therapy in all high-risk ER+, HER2- EBC patients is not cost-effective. However, using MRD detection to justify the addition of abemaciclib treatment dominates standard treatment in this cost-effectiveness analysis. Further evaluation of MRD detection in EBC by means of prospective clinical trials assessing clinical utility is recommended and promising in terms of cost-effectiveness.
Circular RNAs (circRNAs) appear important in tumor progression of colon cancer (CC). We identified an extensive catalog of circRNAs in 181 chemonaive stage I/II colon tumors, who underwent curative ...surgery between 2007 and 2014. We identified circRNAs from RNAseq data, investigated common biology related to circRNA expression, and studied the association between circRNAs and relapse status, tumor stage, consensus molecular subtypes (CMS), tumor localization and microsatellite instability (MSI). We identified 2606 unique circRNAs. 277 circRNAs (derived from 260 genes) were repeatedly occurring in at least 20 patients of which 153 showed a poor or even negative (
< 0.3) correlation with the expression level of their linear gene. The circular junctions for circSATB2, circFGD6, circKMT2C and circPLEKHM3 were validated by Sanger sequencing. Multiple correspondence analysis showed that circRNAs were often co-expressed and that high diversity in circRNAs was associated with favorable disease-free survival (DFS), which was confirmed by Cox regression analysis (Hazard Ratio (HR) 0.60, 95% CI 0.38-0.97,
= 0.036). Considering individual circRNAs, absence of circMGA was significantly associated with relapse, whereas circSATB2, circNAB1, and circCEP192 were associated with both MSI and CMS. This study represents a showcase of the potential clinical utility of circRNAs for prognostic stratification in patients with stage I-II colon cancer and demonstrated that high diversity in circRNAs is associated with favorable DFS.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The identification of patients with high-risk stage II colon cancer who may benefit from adjuvant therapy may allow the clinical approach to be tailored for these patients based on an understanding ...of tumour biology. MicroRNAs have been proposed as markers of the prognosis or treatment response in colorectal cancer. Recently, a 2-microRNA signature (let-7i and miR-10b) was proposed to identify colorectal cancer patients at risk of developing distant metastasis. We assessed the prognostic value of this signature and additional candidate microRNAs in an independent, clinically well-defined, prospectively collected cohort of primary colon cancer patients including stage I-II colon cancer without and stage III colon cancer with adjuvant treatment. The 2-microRNA signature specifically predicted hepatic recurrence in the stage I-II group, but not the overall ability to develop distant metastasis. The addition of miR-30b to the 2-microRNA signature allowed the prediction of both distant metastasis and hepatic recurrence in patients with stage I-II colon cancer who did not receive adjuvant chemotherapy. Available gene expression data allowed us to associate miR-30b expression with axon guidance and let-7i expression with cell adhesion, migration, and motility.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Loss of 3p11-p14 is a frequent event in epithelial cancer and a candidate prognostic biomarker in cervical cancer. In addition to loss, promoter methylation can participate in gene silencing and ...promote tumor aggressiveness. We have performed a complete mapping of promoter methylation at 3p11-p14 in two independent cohorts of cervical cancer patients (n = 149, n = 121), using Illumina 450K methylation arrays. The aim was to investigate whether hyperm-ethylation was frequent and could contribute to gene silencing and disease aggressiveness either alone or combined with loss. By comparing the methylation level of individual CpG sites with corresponding data of normal cervical tissue, 26 out of 41 genes were found to be hypermethylated in both cohorts. The frequency of patients with hypermethylation of these genes was found to be higher at tumor stages of 3 and 4 than in stage 1 tumors. Seventeen of the 26 genes were transcriptionally downregulated in cancer compared to normal tissue, whereof 6 genes showed a significant correlation between methylation and expression. Integrated analysis of methylation, gene dosage, and expression of the 26 hypermethylated genes identified 3 regulation patterns encompassing 8 hypermethylated genes; a methylation driven pattern (C3orf14, GPR27, ZNF717), a gene dosage driven pattern (THOC7, PSMD6), and a combined methylation and gene dosage driven pattern (FHIT, ADAMTS9, LRIG1). In survival analysis, patients with both hypermethylation and loss of LRIG1 had a worse outcome compared to those harboring only hypermethylation or none of the events. C3orf14 emerged as a novel methylation regulated suppressor gene, for which knockdown was found to promote invasive growth in human papilloma virus (HPV)-transformed keratinocytes. In conclusion, hypermethylation at 3p11-p14 is common in cervical cancer and may exert a selection pressure during carcinogenesis alone or combined with loss. Information on both events could lead to improved prognostic markers.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
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