Enamel caries lesion depth obtained by optical coherence tomography and transverse microradiography: a comparative study.

INTRODUCTION: Visual imaging of sub-surface caries lesions is of vital interest in dentistry, which can be obtained by invasive radiography-technique as well as by available non-destructive imaging approaches. Thus, as a first step towards the development of a new innovative approach, the Spectral-domain optical coherence tomography (SD-OCT) was applied to detect the lesion depth in comparison to the established reference technique (transverse microradiography, TMR). METHODS: Bovine enamel specimens were demineralized for 5 days, following previous studies by Braga et al., [2021] and [2022]. For OCT, the resulting artificial lesions were scanned three-dimensionally (SD-OCT), and semiautomated measured (CarLQuant). For TMR, specimens were sectioned and the lesion depth was manually determined (Inspektor Research System). RESULTS: The range of lesion depth detected with OCT was 24.0 to 174.0 µm (mouthrinses study, Braga et al., [2021]), 18.0 to 178.0 µm (toothpastes study) and with TMR 59.2 to 198.0 µm (mouthrinses study), 33.2 to 133.4 µm (toothpastes study, Braga et al., [2022]). We found a strong correlation between both methods in terms of lesion depth (Spearman rankwith outlier p<0.001, Rho=0.75, Spearman rankwithout outlier p=0.001, Rho=0.79). The two methods produce similar results (Passing Bablok regression, 1.16). As deeper is the lesion, the smallest is the difference between both methods as indicated by Bland-Altman-plots. CONCLUSION: Especially in the case of deep lesions, the values obtained by both methods are in agreement, and OCT can potentially substitute TMR to detect and assess lesion depth with the benefit of being non-destructive.

Genomic Considerations in the Treatment of Thyroid Carcinoma.

Highlighting genomically driven targeted therapies to improve outcomes in advanced thyroid carcinoma.

Assessment of the axial resolution of a compact gamma camera with coded aperture collimator.

PURPOSE: Handheld gamma cameras with coded aperture collimators are under investigation for intraoperative imaging in nuclear medicine. Coded apertures are a promising collimation technique for applications such as lymph node localization due to their high sensitivity and the possibility of 3D imaging. We evaluated the axial resolution and computational performance of two reconstruction methods. METHODS: An experimental gamma camera was set up consisting of the pixelated semiconductor detector Timepix3 and MURA mask of rank 31 with round holes of 0.08 mm in diameter in a 0.11 mm thick Tungsten sheet. A set of measurements was taken where a point-like gamma source was placed centrally at 21 different positions within the range of 12-100 mm. For each source position, the detector image was reconstructed in 0.5 mm steps around the true source position, resulting in an image stack. The axial resolution was assessed by the full width at half maximum (FWHM) of the contrast-to-noise ratio (CNR) profile along the z-axis of the stack. Two reconstruction methods were compared: MURA Decoding and a 3D maximum likelihood expectation maximization algorithm (3D-MLEM). RESULTS: While taking 4400 times longer in computation, 3D-MLEM yielded a smaller axial FWHM and a higher CNR. The axial resolution degraded from 5.3 mm and 1.8 mm at 12 mm to 42.2 mm and 13.5 mm at 100 mm for MURA Decoding and 3D-MLEM respectively. CONCLUSION: Our results show that the coded aperture enables the depth estimation of single point-like sources in the near field. Here, 3D-MLEM offered a better axial resolution but was computationally much slower than MURA Decoding, whose reconstruction time is compatible with real-time imaging.

Tooth-composite bond failure with a universal and an etch-and-rinse adhesive depending on mode and frequency of application.

OBJECTIVES: To evaluate the effect of an additional layer of universal adhesive on the interfacial enamel/dentin-composite gap formation in relation to application mode and aging, via spectral domain optical coherence tomography (SD-OCT) and scanning electron microscopy (SEM). METHODS: In vitro class V cavities in 114 caries-free premolars were restored by applying one or two layers of a universal adhesive (Scotchbond Universal, SBU) in self-etch (se) and etch-and-rinse (er) mode or the reference adhesive OptiBond FL (OFL-er). The restorations were imaged by SD-OCT (six groups, n = 8) and SEM (n = 3) directly after filling (t1), water storage (t2, 24 h), embedding (t3), and thermo-mechanical loading (t4, TCML). The interfacial gaps were quantified using 26 parameters and analyzed using principal component analysis and linear mixed effect models. RESULTS: Gap formation at enamel and dentin was significantly influenced by the adhesive, the application mode and number of layers (p < 0.001). This was due to the influence of the SBU-er mode (p < 1e-05), which showed significantly more gap formation and a greater range of variation with double application when compared to SBU-se and OFL. The fewest interfacial gaps occurred with one or two applications of OFL-er and one layer of SBU-er. SIGNIFICANCE: Adhesive application mode and the number of adhesive layers are relevant factors in the tooth-composite bond failure. Double application worsened the adaptation of SBU to freshly prepared dentin conditioned with phosphoric acid.

Evaluation of anti-Xa levels in patients with venous thromboembolism within the first 48 h of anticoagulation with unfractionated heparin.

Background: A 2019 study by Prucnal and colleagues found that the majority of patients treated with unfractionated heparin for pulmonary embolism did not maintain therapeutic activated partial thromboplastin time levels during the first 48 h of therapy. Objective: The purpose of this study was to evaluate the ability of an institution's unfractionated heparin dosing protocol to achieve and maintain therapeutic anti-Xa levels within the first 48 h of therapy in patients with venous thromboembolism. Methods: This retrospective study included 205 patients from May 2016 through September 2020. Patients were divided into two cohorts: bolus plus infusion (N = 89) and infusion only (N = 116). The primary objective was to determine the number of patients who achieved at least one therapeutic level. Results: Overall, 200 patients (97.6%) had at least one therapeutic level with no statistically significant difference between cohorts (p = 0.65). No more than 60% of patients achieved a therapeutic level at any of the 6-h intervals throughout the timeframe. The median time to the first therapeutic level in the overall group was 12.8 h with no statistically significant difference between the bolus plus infusion and infusion-only cohorts (13.3 h versus 12.7 h, respectively, p = 0.48). Conclusions: Most patients were able to achieve at least one therapeutic level within the first 48 h, but fewer were able to maintain therapeutic levels. Further studies are warranted to determine whether alternative dosing strategies would yield consistent achievement of therapeutic levels and affect patient-oriented outcomes.

A phase 1 evaluation of the safety and tolerability of niraparib in combination with everolimus in advanced ovarian and breast cancers.

OBJECTIVES: Phase 1 trial to determine the safety and tolerability of everolimus and niraparib in patients with advanced ovarian and breast malignancies. RESULTS: Fourteen heavily pretreated patients were enrolled (12 high-grade serous ovarian cancer, 1 clear cell ovarian cancer, and 1 triple negative breast cancer). All patients were PARP naïve and received comprehensive genomic profiling prior to enrollment. Two DLTs were experienced in cohort 2 (niraparib 200 mg daily and everolimus 5 mg 3 days per week) with one patient experiencing prolonged thrombocytopenia and the other experiencing severe hypertension. Four additional patients were enrolled after dose de-escalation with one patient again experiencing severe hypertension leading to conclusion of the study. The most frequent grade 3 or greater adverse events were thrombocytopenia, hypertension, anemia, fatigue, neutropenia, and elevated alkaline phosphatase. Two patients had a PR and five patients had SD. ORR was 18% and the CBR was 45% in 11 evaluable patients. Median PFS was 6 months, and median OS is approximately 18 months with three patients still alive at the data cutoff. CONCLUSIONS: The combination of everolimus and niraparib demonstrated significant toxicity at lower doses and is not feasible due to rapid onset and severe hypertension. This limitation possibly blunted the efficacy of the combination as PFS was modest, but OS was surprisingly robust due to three patients with ovarian cancer remaining alive with platinum refractory disease. Further investigation of multiagent blockade of the PI3K pathway combined with PARP is warranted.

Combining Solid and Liquid Biopsy for Therapy Monitoring in Esophageal Cancer.

Esophageal cancer (EC) has one of the highest mortality rates among cancers, making it imperative that therapies are optimized and dynamically adapted to individuals. In this regard, liquid biopsy is an increasingly important method for residual disease monitoring. However, conflicting detection rates (14% versus 60%) and varying cell-free circulating tumor DNA (ctDNA) levels (0.07% versus 0.5%) have been observed in previous studies. Here, we aim to resolve this discrepancy. For 19 EC patients, a complete set of cell-free DNA (cfDNA), formalin-fixed paraffin-embedded tumor tissue (TT) DNA and leukocyte DNA was sequenced (139 libraries). cfDNA was examined in biological duplicates and/or longitudinally, and TT DNA was examined in technical duplicates. In baseline cfDNA, mutations were detected in 12 out of 19 patients (63%); the median ctDNA level was 0.4%. Longitudinal ctDNA changes were consistent with clinical presentation. Considerable mutational diversity was observed in TT, with fewer mutations in cfDNA. The most recurrently mutated genes in TT were TP53, SMAD4, TSHZ3, and SETBP1, with SETBP1 being reported for the first time. ctDNA in blood can be used for therapy monitoring of EC patients. However, a combination of solid and liquid samples should be used to help guide individualized EC therapy.

Phase I dose escalation study of dual PI3K/mTOR inhibition by Sapanisertib and Serabelisib in combination with paclitaxel in patients with advanced solid tumors.

BACKGROUND: Phase I trial to determine the safety and efficacy of paclitaxel, sapanisertib, and serabelisib. PATIENTS AND METHODS: Patients with previously treated advanced solid tumors were eligible for this open label, cohort study of sapanisertib (TAK-228) and serabelisib (TAK-117) with weekly paclitaxel. A traditional 3 + 3 dose escalation design with 5 dosing cohorts was used. Patient reported outcomes were also evaluated. RESULTS: 19 heavily pretreated patients were enrolled (10 ovarian, 3 breast, and 6 endometrial cancers). All patients received comprehensive genomic profiling prior to enrollment. RP2D is sapanisertib 3 or 4 mg, serabelisib 200 mg on days 2-4, 9-11, 16-18 and 23-25 with paclitaxel 80 mg/m2 on days 1, 8 and 15 every 28 days. All patients in Cohort 5 required dose reductions and one patient experienced a DLT. The most frequent grade 3 or 4 adverse events were decreased WBCs (20%), nonfebrile neutropenia (12%), anemia (9%), elevated liver enzymes (4%), and hyperglycemia (11%). 3 patients had a CR, 4 had a PR, and 4 patients had SD > six months. ORR was 47% and CBR was 73% in 15 evaluable patients. Including all 19 enrolled patients, the PFS was 11 months and OS is still ongoing at 17 months. CONCLUSIONS: The combination of sapanisertib, serabelisib, and paclitaxel was safe and generally well tolerated. Preliminary efficacy was remarkable in an area of unmet need, especially for patient with PI3K/AKT/mTOR pathway aberrations. Positive effects and sustained clinical benefit were even seen in patients that were refractory to platinum and had failed taxane, everolimus, or temsirolimus. CLINICAL TRIAL NUMBER: ClinicalTrials.gov, NCT03154294.

Multiscale heterogeneity in gastric adenocarcinoma evolution is an obstacle to precision medicine.

BACKGROUND: Cancer is a somatic evolutionary disease and adenocarcinomas of the stomach and gastroesophageal junction (GC) may serve as a two-dimensional model of cancer expansion, in which tumor subclones are not evenly mixed during tumor progression but rather spatially separated and diversified. We hypothesize that precision medicine efforts are compromised when clinical decisions are based on a single-sample analysis, which ignores the mechanisms of cancer evolution and resulting intratumoral heterogeneity. Using multiregional whole-exome sequencing, we investigated the effect of somatic evolution on intratumoral heterogeneity aiming to shed light on the evolutionary biology of GC. METHODS: The study comprised a prospective discovery cohort of 9 and a validation cohort of 463 GCs. Multiregional whole-exome sequencing was performed using samples form 45 primary tumors and 3 lymph node metastases (range 3-10 tumor samples/patient) of the discovery cohort. RESULTS: In total, the discovery cohort harbored 16,537 non-synonymous mutations. Intratumoral heterogeneity of somatic mutations and copy number variants were present in all tumors of the discovery cohort. Of the non-synonymous mutations, 53-91% were not present in each patient's sample; 399 genes harbored 2-4 different non-synonymous mutations in the same patient; 175 genes showed copy number variations, the majority being heterogeneous, including CD274 (PD-L1). Multi-sample tree-based analyses provided evidence for branched evolution being most complex in a microsatellite instable GC. The analysis of the mode of evolution showed a high degree of heterogeneity in deviation from neutrality within each tumor. We found evidence of parallel evolution and evolutionary trajectories: different mutations of SMAD4 aligned with different subclones and were found only in TP53 mutant GCs. CONCLUSIONS: Neutral and non-neutral somatic evolution shape the mutational landscape in GC along its lateral expansions. It leads to complex spatial intratumoral heterogeneity, where lymph node metastases may stem from different areas of the primary tumor, synchronously. Our findings may have profound effects on future patient management. They illustrate the risk of mis-interpreting tumor genetics based on single-sample analysis and open new avenues for an evolutionary classification of GC, i.e., the discovery of distinct evolutionary trajectories which can be utilized for precision medicine.

The Effect of Solutions Containing Extracts of Vochysia tucanorum Mart., Myrcia bellaCambess., Matricaria chamomilla L. and Malva sylvestris L. on Cariogenic Bacterial Species and Enamel Caries Development.

This study evaluated the effect of experimental solutions containing plant extracts on bacterial species and enamel caries prevention. Microcosm biofilm was produced from human saliva mixed with McBain saliva (0.2% sucrose) on bovine enamel for 5 days (3 days under anaerobiosis and 2 days under aerobiosis) at 37°C. From the 2nd day, the following treatments were applied (1 × 60 s/day): Vochysia tucanorum (10 mg/mL); Myrcia bella (5 mg/mL); Matricaria chamomilla (80 mg/mL); Malva sylvestris, fluoride, and xylitol (Malvatricin Plus®); 0.12% chlorhexidine (CHX, PerioGard®); and PBS (negative control). The medium pH was measured. Quantitative polymerase chain reaction was performed for the detection of Streptococcus mutans and Lactobacillus spp. Enamel demineralization was measured by spectral-domain optical coherence tomography. The data were compared by means of the Kruskal-Wallis/Dunn, two-way ANOVA/Bonferroni, and ANOVA/Tukey tests (p < 0.05). The pH decreased after sucrose exposure; only CHX reestablished pH >5.5 by the last day. CHX also eliminated Lactobacillusspp., but the other treatments did not differ significantly from PBS. Malvatricin Plus® and CHX eliminated S. mutans, but the other treatments did not differ from PBS. Similar results were seen concerning the reduction of lesion depth and reflectivity. The experimental natural-extract solutions were ineffective against cariogenic bacteria and in preventing the development of enamel caries.

Myelin development in visual scene-network tracts beyond late childhood: A multimethod neuroimaging study.

The visual scene-network-comprising the parahippocampal place area (PPA), retrosplenial cortex (RSC), and occipital place area (OPA)-shows a prolonged functional development. Structural development of white matter that underlies the scene-network has not been investigated despite its potential influence on scene-network function. The key factor for white matter maturation is myelination. However, research on myelination using the gold standard method of post-mortem histology is scarce. In vivo alternatives diffusion-weighted imaging (DWI) and myelin water imaging (MWI) so far report broad-scale findings that prohibit inferences concerning the scene-network. Here, we combine MWI, DWI tractography, and fMRI to investigate myelination in scene-network tracts in middle childhood, late childhood, and adulthood. We report increasing myelin from middle childhood to adulthood in right PPA-OPA, and trends towards increases in the left and right RSC-OPA tracts. Investigating tracts to regions highly connected with the scene-network, such as early visual cortex and the hippocampus, did not yield any significant age group differences. Our findings indicate that structural development coincides with functional development in the scene-network, possibly enabling structure-function interactions.

Case report: 16-yr life history and genomic evolution of an ER+ HER2- breast cancer.

Metastatic breast cancer is one of the leading causes of cancer-related death in women. Limited studies have been done on the genomic evolution between primary and metastatic breast cancer. We reconstructed the genomic evolution through the 16-yr history of an ER+ HER2- breast cancer patient to investigate molecular mechanisms of disease relapse and treatment resistance after long-term exposure to hormonal therapy. Genomic and transcriptome profiling was performed on primary breast tumor (2002), initial recurrence (2012), and liver metastasis (2015) samples. Cell-free DNA analysis was performed at 11 time points (2015-2017). Mutational analysis revealed a low mutational burden in the primary tumor that doubled at the time of progression, with driver mutations in PI3K-Akt and RAS-RAF signaling pathways. Phylogenetic analysis showed an early branching off between primary tumor and metastasis. Liquid biopsies, although initially negative, started to detect an ESR1 E380Q mutation in 2016 with increasing allele frequency until the end of 2017. Transcriptome analysis revealed 721 (193 up, 528 down) genes to be differentially expressed between primary tumor and first relapse. The most significantly down-regulated genes were TFF1 and PGR, indicating resistance to aromatase inhibitor (AI) therapy. The most up-regulated genes included PTHLH, S100P, and SOX2, promoting tumor growth and metastasis. This phylogenetic reconstruction of the life history of a single patient's cancer as well as monitoring tumor progression through liquid biopsies allowed for uncovering the molecular mechanisms leading to initial relapse, metastatic spread, and treatment resistance.

Head motion during fMRI tasks is reduced in children and adults if participants take breaks.

Head motion remains a challenging confound in functional magnetic resonance imaging (fMRI) studies of both children and adults. Most pediatric neuroimaging labs have developed experience-based, child-friendly standards concerning e.g. the maximum length of a session or the time between mock scanner training and actual scanning. However, it is unclear which factors of child-friendly neuroimaging approaches are effective in reducing head motion. Here, we investigate three main factors including (i) time lag of mock scanner training to the actual scan, (ii) prior scan time, and (iii) task engagement in a dataset of 77 children (aged 6-13) and 64 adults (aged 18-35) using a multilevel modeling approach. In children, distributing fMRI data acquisition across multiple same-day sessions reduces head motion. In adults, motion is reduced after inside-scanner breaks. Despite these positive effects of splitting up data acquisition, motion increases over the course of a study as well as over the course of a run in both children and adults. Our results suggest that splitting up fMRI data acquisition is an effective tool to reduce head motion in general. At the same time, different ways of splitting up data acquisition benefit children and adults.

Stage IV Colorectal Cancer Patients with High Risk Mutation Profiles Survived 16 Months Longer with Individualized Therapies.

Personalized treatment vs. standard of care is much debated, especially in clinical practice. Here we investigated whether overall survival differences in metastatic colorectal cancer patients are explained by tumor mutation profiles or by treatment differences in real clinical practice. Our retrospective study of metastatic colorectal cancer patients of confirmed European ancestry comprised 54 Americans and 54 gender-matched Germans. The Americans received standard of care, and on treatment failure, 35 patients received individualized treatments. The German patients received standard of care only. Tumor mutations, tumor mutation burden and microsatellite status were identified by using the FoundationOne assay or the IDT Pan-Cancer assay. High-risk patients were identified according to the mutational classification by Schell and colleagues. Results: Kaplan-Meier estimates show the high-risk patients to survive 16 months longer under individualized treatments than those under only standard of care, in the median (p < 0.001). Tumor mutation profiles stratify patients by risk groups but not by country. Conclusions: High-risk patients appear to survive significantly longer (p < 0.001) if they receive individualized treatments after the exhaustion of standard of care treatments. Secondly, the tumor mutation landscape in Americans and Germans is congruent and thus warrants the transatlantic exchange of successful treatment protocols and the harmonization of guidelines.

A Small Hypoxia Signature Predicted pCR Response to Bevacizumab in the Neoadjuvant GeparQuinto Breast Cancer Trial.

PURPOSE: In breast cancer, bevacizumab increased pCR rate but not long-term survival and no predictive markers are available to identify patients with long-term benefit from the drug. EXPERIMENTAL DESIGN: We profiled 289 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) biopsies of HER2-negative patients from the GeparQuinto trial of neoadjuvant chemotherapy ± bevacizumab by exome-capture RNA-sequencing (RNA-seq). In a prospectively planned study, we tested molecular signatures for response prediction. IHC validation was performed using tissue microarrays. RESULTS: We found strong agreement of molecular and pathologic parameters as hormone receptors, grading, and lymphocyte infiltration in 221 high-quality samples. Response rates (49.3% pCR overall) were higher in basal-like (68.9%) and HER2-enriched (45.5%) than in luminal B (35.7%), luminal A (17.9%), and normal-like (20.0%) subtypes. T-cell (OR = 1.60; 95% confidence interval, 1.21-2.12; P = 0.001), proliferation (OR = 2.88; 95% CI, 2.00-4.15; P < 0.001), and hypoxia signatures (OR = 1.92; 95% CI, 1.41-2.60; P < 0.001) significantly predicted pCR in univariate analysis. In a prespecified multivariate logistic regression, a small hypoxia signature predicted pCR (OR = 2.40; 95% CI, 1.28-4.51; P = 0.006) with a significant interaction with bevacizumab treatment (P = 0.020). IHC validation using NDRG1 as marker revealed highly heterogenous expression within tissue leading to profound loss of sensitivity in TMA analysis, still a significant predictive value for pCR was detected (P = 0.025). CONCLUSIONS: Exome-capture RNA-seq characterizes small FFPE core biopsies by reliably detecting factors as for example ER status, grade, and tumor-infiltrating lymphocytes levels. Beside molecular subtypes and immune signatures, a small hypoxia signature predicted pCR to bevacizumab, which could be validated by IHC. The signature can have important applications for bevacizumab treatment in different cancer types and might also have a role for novel combination therapies of bevacizumab with immune checkpoint inhibition.

Prolonged functional development of the parahippocampal place area and occipital place area.

Successful navigation of our surroundings is of high environmental relevance and involves processing of the visual scenery. Scene-processing undergoes a major behavioral improvement during childhood. However, possible neural changes that underlie this cognitive development in scene perception are understudied in comparison to other stimulus categories. We used a functional magnetic resonance imaging (fMRI) scene localizer and behavioral recognition and memory tasks in 7-8-year-olds, 11-12-year-olds, and adults to test whether scene-selective areas-the parahippocampal place area (PPA), the retrosplenial cortex (RSC), and the occipital place area (OPA)-show a change in volume and selectivity with age, and whether this change is correlated with behavioral perception and memory performance. We find that children have a smaller PPA and OPA than adults, while the size of RSC does not differ. Furthermore, selectivity for scenes in the PPA and the OPA, but not in the RSC, increases with age. This increase seems to be driven by both increasing responses to preferred stimuli and decreasing responses to non-preferred stimuli. Our findings extend previous knowledge about visual cortex development by unveiling the underlying mechanisms of age-related volume and selectivity increases in the scene network especially elucidating the poorly understood development of the OPA.

RNA based individualized drug selection in breast cancer patients without patient-matched normal tissue.

BACKGROUND: While standard RNA expression tests stratify patients into risk groups, RNA-Seq can guide personalized drug selection based on expressed mutations, fusion genes, and differential expression (DE) between tumor and normal tissue. However, patient-matched normal tissue may be unavailable. Additionally, biological variability in normal tissue and technological biases may confound results. Therefore, we present normal expression reference data for two sequencing methods that are suitable for breast biopsies. RESULTS: We identified breast cancer related and drug related genes that are expressed uniformly across our normal samples. Large subsets of these genes are identical for formalin fixed paraffin embedded samples and fresh frozen samples. Adipocyte signatures were detected in frozen compared to formalin samples, prepared by surgeons and pathologists, respectively. Gene expression confounded by adipocytes was identified using fat tissue samples. Finally, immune repertoire statistics were obtained for healthy breast, tumor and fat tissues. CONCLUSIONS: Our reference data can be used with patient tumor samples that are asservated and sequenced with a matching aforementioned method. Coefficients of variation are given for normal gene expression. Thus, potential drug selection can be based on confidently overexpressed genes and immune repertoire statistics. MATERIALS AND METHODS: Normal expression from formalin and frozen healthy breast tissue samples using Roche Kapa RiboErase (total RNA) (19 formalin, 9 frozen) and Illumina TruSeq RNA Access (targeted RNA-Seq, aka TruSeq RNA Exome) (11 formalin, 1 frozen), and fat tissue (6 frozen Access). Tumor DE using 10 formalin total RNA tumor samples and 1 frozen targeted RNA tumor sample.

Detection of Cross-Sample Contamination in Multiple Myeloma Samples and Sequencing Data.

The increasing applicability and sensitivity of next generation sequencing methods exacerbate one of the main issues in the molecular biology laboratory, namely cross-sample contamination. This type of contamination, which could massively increase the rate of false-positive calls in sequencing experiments, can originate at each step during the processing of multiple myeloma samples, such as CD138-selection of tumor cells, RNA and DNA isolation or the processing of sequencing libraries. Here we describe a Droplet Digital PCR (ddPCR) method and a simple bioinformatic solution for the detection of contamination in patient's samples and derived sequencing data, which are based on the same principle: detection of alternative alleles for single-nucleotide polymorphisms (SNPs) that are homozygous according to the control (germ line) sample.

The multiple myeloma risk allele at 5q15 lowers ELL2 expression and increases ribosomal gene expression.

Recently, we identified ELL2 as a susceptibility gene for multiple myeloma (MM). To understand its mechanism of action, we performed expression quantitative trait locus analysis in CD138+ plasma cells from 1630 MM patients from four populations. We show that the MM risk allele lowers ELL2 expression in these cells (Pcombined = 2.5 × 10-27; ßcombined = -0.24 SD), but not in peripheral blood or other tissues. Consistent with this, several variants representing the MM risk allele map to regulatory genomic regions, and three yield reduced transcriptional activity in plasmocytoma cell lines. One of these (rs3777189-C) co-locates with the best-supported lead variants for ELL2 expression and MM risk, and reduces binding of MAFF/G/K family transcription factors. Moreover, further analysis reveals that the MM risk allele associates with upregulation of gene sets related to ribosome biogenesis, and knockout/knockdown and rescue experiments in plasmocytoma cell lines support a cause-effect relationship. Our results provide mechanistic insight into MM predisposition.

Genetic Predisposition to Multiple Myeloma at 5q15 Is Mediated by an ELL2 Enhancer Polymorphism.

Multiple myeloma (MM) is a malignancy of plasma cells. Genome-wide association studies have shown that variation at 5q15 influences MM risk. Here, we have sought to decipher the causal variant at 5q15 and the mechanism by which it influences tumorigenesis. We show that rs6877329 G > C resides in a predicted enhancer element that physically interacts with the transcription start site of ELL2. The rs6877329-C risk allele is associated with reduced enhancer activity and lowered ELL2 expression. Since ELL2 is critical to the B cell differentiation process, reduced ELL2 expression is consistent with inherited genetic variation contributing to arrest of plasma cell development, facilitating MM clonal expansion. These data provide evidence for a biological mechanism underlying a hereditary risk of MM at 5q15.