Older adults use fewer muscles to overcome perturbations during a seated locomotor task.

Locomotor perturbations provide insights into humans' response to motor errors. We investigated the differences in motor adaptation and muscle cocontraction between young and older adults during perturbed-arm and -leg recumbent stepping. We hypothesized that besides prolonged adaptation due to use-dependent learning, older adults would exhibit greater muscle cocontraction than young adults in response to the perturbations. Perturbations were brief increases in resistance applied during each stride at the extension onset or midextension of the left or right leg. Seventeen young adults and eleven older adults completed four 10-min perturbed stepping tasks. Subjects were instructed to follow a visual pacing cue, step smoothly, and use all their limbs to drive the stepper. Results showed that young and older adults did not decrease their errors with more perturbation experience, and errors did not wash out after perturbations were removed. Interestingly, older adults consistently had smaller motor errors than young adults in response to the perturbations. Older adults used fewer muscles to drive the stepper and had greater cocontraction than young adults. The results suggest that, despite similar motor error responses, young and older adults use distinctive muscle recruitment patterns to perform the motor task. Age-related motor strategies help track motor changes across the human life span and are a baseline for rehabilitation and performance assessment.NEW & NOTEWORTHY Older adults often demonstrate greater cocontraction and motor errors than young adults in response to motor perturbations. We demonstrated that older adults reduced their motor errors more than young adults with brief perturbations during recumbent stepping while maintaining greater muscle cocontraction. In doing so, older adults largely used one muscle pair to drive the stepper, tibialis anterior and soleus, whereas young adults used all muscles. These two muscles are crucial for maintaining upright balance.

Whose Signals Are Being Amplified? Toward a More Equitable Clinical Psychophysiology.

Research using psychophysiological methods holds great promise for refining clinical assessment, identifying risk factors, and informing treatment. Unfortunately, unique methodological features of existing approaches limit inclusive research participation and, consequently, generalizability. This brief overview and commentary provides a snapshot of the current state of representation in clinical psychophysiology, with a focus on the forms and consequences of ongoing exclusion of Black participants. We illustrate issues of inequity and exclusion that are unique to clinical psychophysiology, considering intersections among social constructions of Blackness and biased design of current technology used to measure electroencephalography, skin conductance, and other signals. We then highlight work by groups dedicated to quantifying and addressing these limitations. We discuss the need for reflection and input from a wider variety of stakeholders to develop and refine new technologies, given the risk of further widening disparities. Finally, we provide broad recommendations for clinical psychophysiology research.

Speeding up: Discrete mediolateral perturbations increased self-paced walking speed in young and older adults.

BACKGROUND: In uncertain environments and with increasing age, humans often walk, slower while taking shorter, quicker, and wider steps, reflective of a cautious gait., Understanding when humans opt to use a cautious gait and the differences in gait, strategies used as people age could be examined with perturbations on a self-paced, treadmill that allows participants to adjust their walking speed. Adding varying degrees, of unpredictability, an inherent element of real-world walking, could also improve, understanding of when specific gait strategies are used RESEARCH QUESTION: We investigated how healthy young and older adults adjust their, gait strategies when responding to perturbations of varying unpredictability. We, hypothesized that more unpredictable perturbations would produce more cautious gait, strategies and be more pronounced in older adults than young adults METHODS: Ten young and ten older adults walked on a self-paced treadmill with, discrete mediolateral treadmill shift perturbations. We changed the shift magnitude, and/or the timing of the perturbations during the gait cycle to vary perturbation, unpredictability. We analyzed walking speed and step kinematics from treadmill and, motion capture data RESULTS: Surprisingly, participants walked faster, not slower, for the conditions with, perturbations. Even more surprising, older adults walked faster overall than young, adults. As expected, participants took faster and wider steps for the most unpredictable, perturbation but also took longer steps, which was not expected. Step kinematic, variability and average step width also increased as perturbation unpredictability, increased, suggesting that the more unpredictable conditions demanded greater, balance control. Additionally, older adults had greater step kinematic variability, highlighted further using detrended step length variability, compared to young adults SIGNIFICANCE: Overall, these findings provide new insights about gait strategies and, suggest that perturbations such as discrete mediolateral treadmill shifts can potentially, be designed to encourage participants to walk faster, if it is beneficial.

Racial disparities in EEG research and their implications for our understanding of the maternal brain.

Racial disparities in maternal health are alarming and persistent. Use of electroencephalography (EEG) and event-related potentials (ERPs) to understand the maternal brain can improve our knowledge of maternal health by providing insight into mechanisms underlying maternal well-being, including implications for child development. However, systematic racial bias exists in EEG methodology-particularly for Black individuals-and in psychological and health research broadly. This paper discusses these biases in the context of EEG/ERP research on the maternal brain. First, we assess the racial/ethnic diversity of existing ERP studies of maternal neural responding to infant/child emotional expressions, using papers from a recent meta-analysis, finding that the majority of mothers represented in this research are of White/European ancestry and that the racially and ethnically diverse samples that are present are limited in terms of geography. Therefore, our current knowledge base in this area may be biased and not generalizable across racially diverse mothers. We outline factors underlying this problem, beginning with the racial bias in EEG equipment that systematically excludes individuals of African descent, and also considering factors specific to research with mothers. Finally, we highlight recent innovations to EEG hardware to better accommodate diverse hairstyles and textures, and other important steps to increase racial and ethnic representativeness in EEG/ERP research with mothers. We urge EEG/ERP researchers who study the maternal brain-including our own research group-to take action to increase racial diversity so that this research area can confidently inform understanding of maternal health and contribute to minimizing maternal health disparities.

Mutation-Agnostic Detection of Colorectal Cancer Using Liquid Biopsy-Based Methylation-Specific Signatures.

Detection of methylation patterns in circulating tumor DNA (ctDNA) can offer a novel approach for cancer diagnostics given the unique signature for each tumor type. We developed a next-generation sequencing (NGS)-based assay targeting 32 CpG sites to detect colorectal cancer-specific ctDNA. NGS was performed on bisulfite-converted libraries and status dichotomization was done using median methylation ratios at all targets. We included plasma samples from patients with metastatic colorectal (n = 20) and non-colorectal cancers (n = 8); and healthy volunteers (n = 4). Median methylation ratio was higher in colorectal cancer compared with non-colorectal cancers (P = .001) and normal donors (P = .005). The assay detected ctDNA in 85% of patients with colorectal cancer at a specificity of 92%. Notably, we were able to detect methylated ctDNA in 75% of patients in whom ctDNA was not detected by other methods. Detection of methylated ctDNA was associated with shorter median progression-free survival compared to non-detection (8 weeks versus 54 weeks; P = .027).

Longitudinal Monitoring of Circulating Tumor DNA to Predict Treatment Outcomes in Advanced Cancers.

PURPOSE: The response to cancer therapies is typically assessed with radiologic imaging 6-10 weeks after treatment initiation. Circulating tumor DNA (ctDNA), however, has a short half-life, and dynamic changes in ctDNA quantity may allow for earlier assessment of the therapeutic response. METHODS: Patients with advanced solid tumors referred to the Department of Investigational Cancer Therapeutics at The University of Texas MD Anderson Cancer Center were invited to participate in a liquid biopsy protocol for which serial blood samples were collected before, during, and after systemic therapy. We isolated ctDNA from serially collected plasma samples at baseline, mid-treatment, and first restaging. Genomically informed droplet digital polymerase chain reaction (ddPCR) was performed, and ctDNA quantities were reported as aggregate variant allele frequencies for all detected molecular aberrations. RESULTS: We included 204 patients receiving 260 systemic therapies. The ctDNA detection rate was higher in progressors (patients with progressive disease) compared with nonprogressors (patients with stable disease, partial responses, or complete responses) at all time points (P < .009). Moreover, ctDNA detection was associated with a shorter median time-to-treatment failure (P ≤ .001). Positive delta and slope values for changes in ctDNA quantity were more frequent in progressors (P ≤ .03 and P < .001, respectively) and were associated with a shorter median time-to-treatment failure (P ≤ .014 and P < .001, respectively). Increasing ctDNA quantity was predictive of clinical and/or radiologic progressive disease in 73% of patients (median lead time, 23 days). CONCLUSION: Detection of ctDNA and early dynamic changes in its quantity can predict the clinical outcomes of systemic therapies in patients with advanced solid tumors.

Step-to-step variability indicates disruption to balance control when linking the arms and legs during treadmill walking.

We recently discovered that a rope-pulley system that mechanically coupling the arms, legs and treadmill during walking can assist with forward propulsion in healthy subjects, leading to significant reductions in metabolic cost. However, walking balance may have been compromised, which could hinder the potential use of this device for gait rehabilitation. We performed a secondary analysis by quantifying average step width, step length, and step time, and used their variability to reflect simple metrics of walking balance (n = 8). We predicted an increased variability in at least one of these metrics when using the device, which would indicate disruptions to walking balance. When walking with the device, subjects increased their average step width (p < 0.05), but variability in step width and step length remained similar (p's > 0.05). However, the effect size for step length variability when compared to that of mechanical perturbation experiments suggest a minimal to moderate disruption in balance (Rosenthal ES = 0.385). The most notable decrement in walking balance was an increase in step time variability (p < 0.05; Cohen's d = 1.286). Its effect size reveals a moderate disruption when compared to the effect sizes observed in those with balance deficits (effect sizes ranged between 0.486 to 1.509). Overall, we conclude that healthy subjects experienced minimal to moderate disruptions in walking balance when using with this device. These data indicate that in future clinical experiments, it will be important to not only consider the mechanical and metabolic effects of using such a device but also its potential to disrupt walking balance, which may be exacerbated in patients with poor balance control.

Monitoring of Dynamic Changes and Clonal Evolution in Circulating Tumor DNA From Patients With IDH-Mutated Cholangiocarcinoma Treated With Isocitrate Dehydrogenase Inhibitors.

PURPOSE: IDH mutations occur in about 30% of patients with cholangiocarcinoma. Analysis of mutations in circulating tumor DNA (ctDNA) can be performed by droplet digital polymerase chain reaction (ddPCR). The analysis of ctDNA is a feasible approach to detect IDH mutations. METHODS: We isolated ctDNA from the blood of patients with IDH-mutated advanced cholangiocarcinoma collected at baseline, on therapy, and at progression to isocitrate dehydrogenase (IDH) inhibitors. RESULTS: Of 31 patients with IDH1R132 (n = 26) or IDH2R172 mutations (n = 5) in the tumor, IDH mutations were detected in 84% of ctDNA samples analyzed by ddPCR and in 83% of ctDNA samples analyzed by next-generation sequencing (NGS). Patients with a low variant allele frequency of ctDNA detected by NGS at baseline had a longer median time to treatment failure compared to patients with high variant allele frequency of ctDNA (3.6 v 1.5 months; P = .008). Patients with a decrease in IDH-mutated ctDNA on therapy by ddPCR compared with no change/increase had a trend to a longer median survival (P = .07). Most frequent emergent alterations in ctDNA by NGS at progression were ARID1A (n = 3) and TP53 mutations (n = 3). CONCLUSION: Detection of IDH mutations in ctDNA in patients with advanced cholangiocarcinoma is feasible, and dynamic changes in ctDNA can correspond with the clinical course and clonal evolution.

Small directional treadmill perturbations induce differential gait stability adaptation.

Introducing unexpected perturbations to challenge gait stability is an effective approach to investigate balance control strategies. Little is known about the extent to which people can respond to small perturbations during walking. This study aimed to determine how subjects adapted gait stability to multidirectional perturbations with small magnitudes applied on a stride-by-stride basis. Ten healthy young subjects walked on a treadmill that either briefly decelerated belt speed ("stick"), accelerated belt speed ("slip"), or shifted the platform medial-laterally at right leg mid-stance. We quantified gait stability adaptation in both anterior-posterior and medial-lateral directions using margin of stability and its components, base of support, and extrapolated center of mass. Gait stability was disrupted upon initially experiencing the small perturbations as margin of stability decreased in the stick, slip, and medial shift perturbations and increased in the lateral shift perturbation. Gait stability metrics were generally disrupted more for perturbations in the coincident direction. Subjects employed both feedback and feedforward strategies in response to the small perturbations, but mostly used feedback strategies during adaptation. Subjects primarily used base of support (foot placement) control in the lateral shift perturbation and extrapolated center of mass control in the slip and medial shift perturbations. These findings provide new knowledge about the extent of gait stability adaptation to small magnitude perturbations applied on a stride-by-stride basis and reveal potential new approaches for balance training interventions to target foot placement and center of mass control.NEW & NOTEWORTHY Little is known about if and how humans can adapt to small magnitude perturbations experienced on a stride-by-stride basis during walking. Here, we show that even small perturbations disrupted gait stability and that subjects could still adapt their reactive balance control. Depending on the perturbation direction, subjects might prefer adjusting their foot placement over their center of mass and vice versa. These findings could help potentially tune balance training to target specific aspects of balance.

A novel method for liquid-phase extraction of cell-free DNA for detection of circulating tumor DNA.

Low yields of extracted cell-free DNA (cfDNA) from plasma limit continued development of liquid biopsy in cancer, especially in early-stage cancer diagnostics and cancer screening applications. We investigate a novel liquid-phase-based DNA isolation method that utilizes aqueous two-phase systems to purify and concentrate circulating cfDNA. The PHASIFY MAX and PHASIFY ENRICH kits were compared to a commonly employed solid-phase extraction method on their ability to extract cfDNA from a set of 91 frozen plasma samples from cancer patients. Droplet digital PCR (ddPCR) was used as the downstream diagnostic to detect mutant copies. Compared to the QIAamp Circulating Nucleic Acid (QCNA) kit, the PHASIFY MAX method demonstrated 60% increase in DNA yield and 171% increase in mutant copy recovery, and the PHASIFY ENRICH kit demonstrated a 35% decrease in DNA yield with a 153% increase in mutant copy recovery. A follow-up study with PHASIFY ENRICH resulted in the positive conversion of 9 out of 47 plasma samples previously determined negative with QCNA extraction (all with known positive tissue genotyping). Our results indicate that this novel extraction technique offers higher cfDNA recovery resulting in better sensitivity for detection of cfDNA mutations compared to a commonly used solid-phase extraction method.

Speed-related but not detrended gait variability increases with more sensitive self-paced treadmill controllers at multiple slopes.

Self-paced treadmills are being used more frequently to study humans walking with their self-selected gaits on a range of slopes. There are multiple options to purchase a treadmill with a built-in controller, or implement a custom written self-paced controller, which raises questions about how self-paced controller affect treadmill speed and gait biomechanics on multiple slopes. This study investigated how different self-paced treadmill controller sensitivities affected gait parameters and variability on decline, level, and incline slopes. We hypothesized that increasing self-paced controller sensitivity would increase gait variability on each slope. We also hypothesized that detrended variability could help mitigate differences in variability that arise from differences in speed fluctuations created by the self-paced controllers. Ten young adults walked on a self-paced treadmill using three controller sensitivities (low, medium, and high) and fixed speeds at three slopes (decline, -10°; level, 0°; incline, +10°). Within each slope, average walking speeds and spatiotemporal gait parameters were similar regardless of self-paced controller sensitivity. With higher controller sensitivities on each slope, speed fluctuations, speed variance, and step length variance increased whereas step frequency variance and step width variance were unaffected. Detrended variance was not affected by controller sensitivity suggesting that detrending variability helps mitigate differences associated with treadmill speed fluctuations. Speed-trend step length variances, however, increased with more sensitive controllers. Further, detrended step length variances were similar for self-paced and fixed speed walking, whereas self-paced walking included substantial speed-trend step length variance not present in fixed speed walking. In addition, regardless of the self-paced controller, subjects walked fastest on the level slope with the longest steps, narrowest steps, and least variance. Overall, our findings suggest that separating gait variability into speed-trend and detrended variability could be beneficial for interpreting gait variability among multiple self-paced treadmill studies and when comparing self-paced walking with fixed speed walking.

Differential Theta-Band Signatures of the Anterior Cingulate and Motor Cortices During Seated Locomotor Perturbations.

Quantifying motor and cortical responses to perturbations during seated locomotor tasks such as recumbent stepping and cycling will expand and improve the understanding of locomotor adaptation processes beyond just perturbed gait. Using a perturbed recumbent stepping protocol, we hypothesized motor errors and anterior cingulate activity would decrease with time, and perturbation timing would influence electrocortical elicitation. Young adults (n = 17) completed four 10-minute arms and legs stepping tasks, with perturbations applied at every left or right leg extension-onset or mid-extension. A random no-perturbation "catch" stride occurred in every five perturbed strides. We instructed subjects to follow a pacing cue and to step smoothly, and we quantified temporal and spatial motor errors. We used high-density electroencephalography to estimate sources of electrocortical fluctuations shared among >70% of subjects. Temporal and spatial errors did not decrease from early to late for either perturbed or catch strides. Interestingly, spatial errors post-perturbation did not return to pre-perturbation levels, suggesting use-dependent learning occurred. Theta (3-8 Hz) synchronization in the anterior cingulate cortex and left and right supplementary motor areas (SMA) emerged near the perturbation event, and extension-onset perturbations elicited greater theta-band power than mid-extension perturbations. Even though motor errors did not adapt, anterior cingulate theta synchronization decreased from early to late perturbed strides, but only during the right-side tasks. Additionally, SMA mainly demonstrated specialized, not contralateral, lateralization. Overall, seated locomotor perturbations produced differential theta-band responses in the anterior cingulate and SMAs, suggesting that tuning perturbation parameters, e.g., timing, can potentially modify electrocortical responses.

Hormonal modulation of ESR1 mutant metastasis.

Estrogen receptor alpha gene (ESR1) mutations occur frequently in ER-positive metastatic breast cancer, and confer clinical resistance to aromatase inhibitors. Expression of the ESR1 Y537S mutation induced an epithelial-mesenchymal transition (EMT) with cells exhibiting enhanced migration and invasion potential in vitro. When small subpopulations of Y537S ESR1 mutant cells were injected along with WT parental cells, tumor growth was enhanced with mutant cells becoming the predominant population in distant metastases. Y537S mutant primary xenograft tumors were resistant to the antiestrogen tamoxifen (Tam) as well as to estradiol (E2) withdrawal. Y537S ESR1 mutant primary tumors metastasized efficiently in the absence of E2; however, Tam treatment significantly inhibited metastasis to distant sites. We identified a nine-gene expression signature, which predicted clinical outcomes of ER-positive breast cancer patients, as well as breast cancer metastasis to the lung. Androgen receptor (AR) protein levels were increased in mutant models, and the AR agonist dihydrotestosterone significantly inhibited estrogen-regulated gene expression, EMT, and distant metastasis in vivo, suggesting that AR may play a role in distant metastatic progression of ESR1 mutant tumors.

Dose-escalation study of vemurafenib with sorafenib or crizotinib in patients with BRAF-mutated advanced cancers.

BACKGROUND: BRAF inhibitors are effective in melanoma and other cancers with BRAF mutations; however, patients ultimately develop therapeutic resistance through the activation of alternative signaling pathways such as RAF/RAS or MET. The authors hypothesized that combining the BRAF inhibitor vemurafenib with either the multikinase inhibitor sorafenib or the MET inhibitor crizotinib could overcome therapeutic resistance. METHODS: Patients with advanced cancers and BRAF mutations were enrolled in a dose-escalation study (3 + 3 design) to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities (DLTs) of vemurafenib with sorafenib (VS) or vemurafenib with crizotinib (VC). RESULTS: In total, 38 patients (VS, n = 24; VC, n = 14) were enrolled, and melanoma was the most represented tumor type (VS, 38%; VC, 64%). In the VS arm, vemurafenib 720 mg twice daily and sorafenib 400 mg am/200 mg pm were identified as the MTDs, DLTs included grade 3 rash (n = 2) and grade 3 hypertension, and partial responses were reported in 5 patients (21%), including 2 with ovarian cancer who had received previous treatment with BRAF, MEK, or ERK inhibitors. In the VC arm, vemurafenib 720 mg twice daily and crizotinib 250 mg daily were identified as the MTDs, DLTs included grade 3 rash (n = 2), and partial responses were reported in 4 patients (29%; melanoma, n = 3; lung adenocarcinoma, n = 1) who had received previous treatment with BRAF, MEK, and/or ERK inhibitors. Optional longitudinal collection of plasma to assess dynamic changes in circulating tumor DNA demonstrated the elimination of BRAF-mutant DNA from plasma during therapy (P = .005). CONCLUSIONS: Vemurafenib combined with sorafenib or crizotinib was well tolerated with encouraging activity, including among patients who previously received treatment with BRAF, MEK, or ERK inhibitors.

Phase I Study of Everolimus, Letrozole, and Trastuzumab in Patients with Hormone Receptor-positive Metastatic Breast Cancer or Other Solid Tumors.

PURPOSE: Doublets of everolimus with letrozole or trastuzumab have demonstrated activity against HER2-positive breast cancer, suggesting that the triple combination can have synergistic anticancer activity. PATIENTS AND METHODS: This first-in-human dose-escalation study (NCT02152943) enrolled patients with hormone receptor- positive, HER2-positive (defined by amplification, overexpression, or mutation) treatment-refractory advanced cancers to receive escalating doses (3+3 design) of daily oral letrozole (days 1-21), daily oral everolimus (days 1-21), and intravenous trastuzumab (day 1) every 21 days to determine dose-limiting toxicities (DLT) and MTD or recommended phase II dose (RP2D). RESULTS: A total of 32 patients with hormone receptor-positive, HER2-positive (amplification, n = 27; overexpression, n = 1; and mutation, n = 4) advanced breast cancer (n = 26) or other cancers (n = 6) were enrolled. The most frequent grade ≥3 adverse events included hyperglycemia (n = 4), anemia (n = 3), thrombocytopenia (n = 2), and mucositis (n = 2). DLTs included grade 3 mucositis and grade 4 neutropenia, and trastuzumab given as an 8 mg/kg loading dose on day 1 of cycle 1 followed by a 6 mg/kg maintenance dose on day 1 of subsequent cycles plus 10 mg everolimus daily and 2.5 mg letrozole daily every 21 days was declared as RP2D. Five patients with breast cancer (four with HER2 amplification and one with HER2 mutation) had partial responses. HER2 amplification in circulating cell-free DNA at baseline was associated with shorter progression-free and overall survival durations (P < 0.05). CONCLUSIONS: Everolimus, letrozole, and trastuzumab have a favorable safety profile and elicit encouraging signals of anticancer activity in patients with heavily pretreated hormone receptor- and HER2-positive advanced cancers.

Dry Epidermal Electrodes Can Provide Long-Term High Fidelity Electromyography for Limited Dynamic Lower Limb Movements.

Due to the limitations of standard wet Silver/Silver Chloride (Ag/AgCl) hydrogel electrodes and the growing demand for long-term high fidelity surface electromyography (EMG) recording, dry epidermal electrodes are of great interest. Evaluating the usability and signal fidelity of dry epidermal electrodes could help determine the extent of potential applications using EMG electrodes. We collected EMG signals over eight days from the right rectus femoris of seven subjects using single-use dry epidermal electrodes and traditional Ag/AgCl electrodes while covered and uncovered during dynamic movements (leg extension, sit-to-stand, and treadmill walking at 0.75 m/s and 1.30 m/s). We quantified signal fidelity using signal-to-noise ratio (SNR); signal-to-motion ratio (SMR); and a metric we previously developed, the Signal Quality Index, which considers that better EMG signal quality requires both good signal-to-noise ratio and good signal-to-motion ratio. Wear patterns over the eight days degraded EMG signal quality. Uncovered epidermal electrodes that remained intact and maintained good adhesion to the skin had signal-to-noise ratios, signal-to-motion ratios, and Signal Quality Index values that were above the acceptable thresholds for limited dynamic lower limb movements (leg extension and sit-to-stand). This indicated that dry epidermal electrodes could provide good signal quality across all subjects for five days for these movements. For walking, the signal-to-noise ratios of the uncovered epidermal electrodes were still above the acceptable threshold, but signal-to-motion ratios and the Signal Quality Index values were far below the acceptable thresholds. The signal quality of the epidermal electrodes that showed no visible wear was stable over five days. As expected, covering the epidermal electrodes improved signal quality, but only for limited dynamic lower limb movements. Overall, single-use dry epidermal electrodes were able to maintain high signal quality for long-term EMG recording during limited dynamic lower limb movements, but further improvement is needed to reduce motion artifacts for whole body dynamic movements such as walking.

RAS/RAF mutations in tumor samples and cell-free DNA from plasma and bone marrow aspirates in multiple myeloma patients.

Purpose: To evaluate the detection of gene mutations in bone marrow biopsy and circulating free DNA (cfDNA) from plasma in multiple myeloma (MM). Experimental design: We used cell-free DNA from plasma and bone marrow to test BRAF V600, KRAS G12/G13, NRAS G12/G13 and NRAS Q61 mutations using multiplex assays for droplet digital PCR (ddPCR), and evaluated results with clinical outcomes. Results: We found of 83 patients, the detectable mutation frequencies for the above four genes were 4 (5%), 13 (16%), 3 (4%) and 14 (17%) in bone marrow, respectively. The median variant allelic frequency (VAF) in most mutations were 1.595%. In 17 paired cfDNA samples, the detectable mutation frequencies for the above four genes were 5 (30%), 1 (6%), 0 (0%) and 3 (18%) respectively, and the median VAF rate was 2.9%. Agreement between bone marrow DNA and plasma cfDNA were 76%, 100%, 100% and 100% compared to the tissue detections, respectively. In 17 patients with paired bone marrow and plasma samples, the above four mutations were 3 (18%), 1 (6%), 0 (0%) and 2 (12%) respectively, with the agreement rates of 88%, 88%, 100% and 100% compared to tissue detections. Of 57 patients with available outcome data, high mutation VAF had a shorter median survival than patients with low mutation VAF (P=0.0322). Conclusions: Oncogenic mutations in BRAF, KRAS and NRAS genes can be detected in the bone marrow and plasma cfDNA with ddPCR in patients with MM patients and high VAF is associated with short survival.