Can a Liquid Biopsy Detect Circulating Tumor DNA With Low-passage Whole-genome Sequencing in Patients With a Sarcoma? A Pilot Evaluation.
A liquid biopsy is a test that evaluates the status of a disease by analyzing a sample of bodily fluid, most commonly blood. In recent years, there has been progress in the development and clinical application of liquid biopsy methods to identify blood-based, tumor-specific biomarkers for many cancer types. However, the implementation of these technologies to aid in the treatment of patients who have a sarcoma remains behind other fields of cancer medicine. For this study, we chose to evaluate a sarcoma liquid biopsy based on circulating tumor DNA (ctDNA). All human beings have normal cell-free DNA (cfDNA) circulating in the blood. In contrast with cfDNA, ctDNA is genetic material present in the blood stream that is derived from a tumor. ctDNA carries the unique genomic fingerprint of the tumor with changes that are not present in normal circulating cfDNA. A successful ctDNA liquid biopsy must be able to target these tumor-specific genetic alterations. For instance, epidermal growth factor receptor (EGFR) mutations are common in lung cancers, and ctDNA liquid biopsies are currently in clinical use to evaluate the status of disease in patients who have a lung cancer by detecting EGFR mutations in the blood. As opposed to many carcinomas, sarcomas do not have common recurrent mutations that could serve as the foundation to a ctDNA liquid biopsy. However, many sarcomas have structural changes to their chromosomes, including gains and losses of portions or entire chromosomes, known as copy number alterations (CNAs), that could serve as a target for a ctDNA liquid biopsy. Murine double minute 2 (MDM2) amplification in select lipomatous tumors or parosteal osteosarcoma is an example of a CNA due to the presence of extra copies of a segment of the long arm of chromosome 12. Since a majority of sarcomas demonstrate a complex karyotype with numerous CNAs, a blood-based liquid biopsy strategy that searches for these CNAs may be able to detect the presence of sarcoma ctDNA. Whole-genome sequencing (WGS) is a next-generation sequencing technique that evaluates the entire genome. The depth of coverage of WGS refers to how detailed the sequencing is, like higher versus lower power on a microscope. WGS can be performed with high-depth sequencing (that is, > 60×), which can detect individual point mutations, or low-depth sequencing (that is, 0.1× to 5×), referred to as low-passage whole-genome sequencing (LP-WGS), which may not detect individual mutations but can detect structural chromosomal changes including gains and losses (that is, CNAs). While similar strategies have shown favorable early results for specific sarcoma subtypes, LP-WGS has not been evaluated for applicability to the broader population of patients who have a sarcoma.
Does an LP-WGS liquid biopsy evaluating for CNAs detect ctDNA in plasma samples from patients who have sarcomas representing a variety of histologic subtypes?
This was a retrospective study conducted at a community-based, tertiary referral center. Nine paired (plasma and formalin-fixed paraffin-embedded [FFPE] tissue) and four unpaired (plasma) specimens from patients who had a sarcoma were obtained from a commercial biospecimen bank. Three control specimens from individuals who did not have cancer were also obtained. The paired and unpaired specimens from patients who had a sarcoma represented a variety of sarcoma histologic subtypes. cfDNA was extracted, amplified, and quantified. Libraries were prepared, and LP-WGS was performed using a NextSeq 500 next-generation sequencing machine at a low depth of sequencing coverage (∼1×). The ichorCNA bioinformatics algorithm, which was designed to detect CNAs from low-depth genomic sequencing data, was used to analyze the data. In contrast with the gold standard for diagnosis in the form of histopathologic analysis of a tissue sample, this test does not discriminate between sarcoma subtypes but detects the presence of tumor-derived CNAs within the ctDNA in the blood that should not be present in a patient who does not have cancer. The liquid biopsy was positive for the detection of cancer if the ichorCNA algorithm detected the presence of ctDNA. The algorithm was also used to quantitatively estimate the percent ctDNA within the cfDNA. The concentration of ctDNA was then calculated from the percent ctDNA relative to the total concentration of cfDNA. The CNAs of the paired FFPE tissue and plasma samples were graphically visualized using aCNViewer software.
This LP-WGS liquid biopsy detected ctDNA in 9 of 13 of the plasma specimens from patients with a sarcoma. The other four samples from patients with a sarcoma and all serum specimens from patients without cancer had no detectable ctDNA. Of those 9 patients with positive liquid biopsy results, the percent ctDNA ranged from 6% to 11%, and calculated ctDNA quantities were 0.04 to 5.6 ng/mL, which are levels to be expected when ctDNA is detectable.
In this small pilot study, we were able to detect sarcoma ctDNA with an LP-WGS liquid biopsy searching for CNAs in the plasma of most patients who had a sarcoma representing a variety of histologic subtypes.
These results suggest that an LP-WGS liquid biopsy evaluating for CNAs to identify ctDNA may be more broadly applicable to the population of patients who have a sarcoma than previously reported in studies focusing on specific subtypes. Large prospective clinical trials that gather samples at multiple time points during the process of diagnosis, treatment, and surveillance will be needed to further assess whether this technique can be clinically useful. At our institution, we are in the process of developing a large prospective clinical trial for this purpose.
Anderson CJ
，Yang H
，Parsons J
，Ahrens WA
，Jagosky MH
，Hsu JH
，Patt JC
，Kneisl JS
，Steuerwald NM
... -
《-》
RETRACTED: Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial.
Chloroquine and hydroxychloroquine have been found to be efficient on SARS-CoV-2, and reported to be efficient in Chinese COV-19 patients. We evaluate the effect of hydroxychloroquine on respiratory viral loads.
French Confirmed COVID-19 patients were included in a single arm protocol from early March to March 16th, to receive 600mg of hydroxychloroquine daily and their viral load in nasopharyngeal swabs was tested daily in a hospital setting. Depending on their clinical presentation, azithromycin was added to the treatment. Untreated patients from another center and cases refusing the protocol were included as negative controls. Presence and absence of virus at Day6-post inclusion was considered the end point.
Six patients were asymptomatic, 22 had upper respiratory tract infection symptoms and eight had lower respiratory tract infection symptoms. Twenty cases were treated in this study and showed a significant reduction of the viral carriage at D6-post inclusion compared to controls, and much lower average carrying duration than reported in the litterature for untreated patients. Azithromycin added to hydroxychloroquine was significantly more efficient for virus elimination.
Despite its small sample size, our survey shows that hydroxychloroquine treatment is significantly associated with viral load reduction/disappearance in COVID-19 patients and its effect is reinforced by azithromycin.
This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/locate/withdrawalpolicy). Concerns have been raised regarding this article, the substance of which relate to the articles' adherence to Elsevier's publishing ethics policies and the appropriate conduct of research involving human participants, as well as concerns raised by three of the authors themselves regarding the article's methodology and conclusions. Elsevier's Research Integrity and Publishing Ethics Team, in collaboration with the journal's co-owner, the International Society of Antimicrobial Chemotherapy (ISAC), and with guidance from an impartial field expert acting in the role of an independent Publishing Ethics Advisor, Dr. Jim Gray, Consultant Microbiologist at the Birmingham Children's and Women's Hospitals, U.K., conducted an investigation and determined that the below points constituted cause for retraction: • The journal has been unable to confirm whether any of the patients for this study were accrued before ethical approval had been obtained. The ethical approval dates for this article are stated as being 5th and 6th of March 2020 (ANSM and CPP respectively), while the article states that recruitment began in “early March”. The 17th author, Prof. Philippe Brouqui, has confirmed that the start date for patient accrual was 6th March 2020. The journal has not been able to establish whether all patients could have entered into the study in time for the data to have been analysed and included in the manuscript prior to its submission on the 20th March 2020, nor whether all patients were enrolled in the study upon admission as opposed to having been hospitalised for some time before starting the treatment described in the article. Additionally, the journal has not been able to establish whether there was equipoise between the study patients and the control patients. • The journal has not been able to establish whether the subjects in this study should have provided informed consent to receive azithromycin as part of the study. The journal has concluded that that there is reasonable cause to conclude that azithromycin was not considered standard care at the time of the study. The 17th author, Prof. Philippe Brouqui has attested that azithromycin treatment was not, at the time of the study, an experimental treatment but a possible treatment for, or preventative measure against, bacterial superinfections of viral pneumonia as described in section 2.4 of the article, and as such the treatment should be categorised as standard care that would not require informed consent. This does not fully address the journal's concerns around the use of azithromycin in the study. In section 3.1 of the article, it is stated that six patients received azithromycin to prevent (rather than treat) bacterial superinfection. All of these were amongst the patients who also received hydroxychloroquine (HCQ). None of the control patients are reported to have received azithromycin. This would indicate that only patients in the HCQ arm received azithromycin, all of whom were in one center. The recommendations for use of macrolides in France at the time the study was conducted indicate that azithromycin would not have been a logical agent to use as first-line prophylaxis against pneumonia due to the frequency of macrolide resistance amongst bacteria such as pneumococci. These two points suggest that azithromycin would not have been standard practice across southern France at the time the study was conducted and would have required informed consent. • Three of the authors of this article, Dr. Johan Courjon, Prof. Valérie Giordanengo, and Dr. Stéphane Honoré have contacted the journal to assert their opinion that they have concerns regarding the presentation and interpretation of results in this article and have stated they no longer wish to see their names associated with the article. • Author Prof. Valérie Giordanengo informed the journal that while the PCR tests administered in Nice were interpreted according to the recommendations of the national reference center, it is believed that those carried out in Marseille were not conducted using the same technique or not interpreted according to the same recommendations, which in her opinion would have resulted in a bias in the analysis of the data. This raises concerns as to whether the study was partially conducted counter to national guidelines at that time. The 17th author, Prof. Philippe Brouqui has attested that the PCR methodology was explained in reference 17 of the article. However, the article referred to by reference 17 describes several diagnostic approaches that were used (one PCR targeting the envelope protein only; another targeting the spike protein; and three commercially produced systems by QuantiNova, Biofire, and FTD). This reference does not clarify how the results were interpreted. It has also been noted during investigation of these concerns that only 76% (19/25) of patients were viral culture positive, resulting in uncertainty in the interpretation of PCR reports as has been raised by Prof. Giordanengo. As part of the investigation, the corresponding author was contacted and asked to provide an explanation for the above concerns. No response has been received within the deadline provided by the journal. Responses were received by the 3rd and 17th authors, Prof. Philippe Parola and Prof. Philippe Brouqui, respectively, and were reviewed as part of the investigation. These two authors, in addition to 1st author Dr. Philippe Gautret, 13th author Prof. Philippe Colson, and 15th author Prof. Bernard La Scola, disagreed with the retraction and dispute the grounds for it. Having followed due process and concluded the aforementioned investigation and based on the recommendation of Dr. Jim Gray acting in his capacity as independent Publishing Ethics Advisor, the co-owners of the journal (Elsevier and ISAC) have therefore taken the decision to retract the article.
Gautret P
，Lagier JC
，Parola P
，Hoang VT
，Meddeb L
，Mailhe M
，Doudier B
，Courjon J
，Giordanengo V
，Vieira VE
，Tissot Dupont H
，Honoré S
，Colson P
，Chabrière E
，La Scola B
，Rolain JM
，Brouqui P
，Raoult D
... -
《-》
Comparison of Two Modern Survival Prediction Tools, SORG-MLA and METSSS, in Patients With Symptomatic Long-bone Metastases Who Underwent Local Treatment With Surgery Followed by Radiotherapy and With Radiotherapy Alone.
Survival estimation for patients with symptomatic skeletal metastases ideally should be made before a type of local treatment has already been determined. Currently available survival prediction tools, however, were generated using data from patients treated either operatively or with local radiation alone, raising concerns about whether they would generalize well to all patients presenting for assessment. The Skeletal Oncology Research Group machine-learning algorithm (SORG-MLA), trained with institution-based data of surgically treated patients, and the Metastases location, Elderly, Tumor primary, Sex, Sickness/comorbidity, and Site of radiotherapy model (METSSS), trained with registry-based data of patients treated with radiotherapy alone, are two of the most recently developed survival prediction models, but they have not been tested on patients whose local treatment strategy is not yet decided.
(1) Which of these two survival prediction models performed better in a mixed cohort made up both of patients who received local treatment with surgery followed by radiotherapy and who had radiation alone for symptomatic bone metastases? (2) Which model performed better among patients whose local treatment consisted of only palliative radiotherapy? (3) Are laboratory values used by SORG-MLA, which are not included in METSSS, independently associated with survival after controlling for predictions made by METSSS?
Between 2010 and 2018, we provided local treatment for 2113 adult patients with skeletal metastases in the extremities at an urban tertiary referral academic medical center using one of two strategies: (1) surgery followed by postoperative radiotherapy or (2) palliative radiotherapy alone. Every patient's survivorship status was ascertained either by their medical records or the national death registry from the Taiwanese National Health Insurance Administration. After applying a priori designated exclusion criteria, 91% (1920) were analyzed here. Among them, 48% (920) of the patients were female, and the median (IQR) age was 62 years (53 to 70 years). Lung was the most common primary tumor site (41% [782]), and 59% (1128) of patients had other skeletal metastases in addition to the treated lesion(s). In general, the indications for surgery were the presence of a complete pathologic fracture or an impending pathologic fracture, defined as having a Mirels score of ≥ 9, in patients with an American Society of Anesthesiologists (ASA) classification of less than or equal to IV and who were considered fit for surgery. The indications for radiotherapy were relief of pain, local tumor control, prevention of skeletal-related events, and any combination of the above. In all, 84% (1610) of the patients received palliative radiotherapy alone as local treatment for the target lesion(s), and 16% (310) underwent surgery followed by postoperative radiotherapy. Neither METSSS nor SORG-MLA was used at the point of care to aid clinical decision-making during the treatment period. Survival was retrospectively estimated by these two models to test their potential for providing survival probabilities. We first compared SORG to METSSS in the entire population. Then, we repeated the comparison in patients who received local treatment with palliative radiation alone. We assessed model performance by area under the receiver operating characteristic curve (AUROC), calibration analysis, Brier score, and decision curve analysis (DCA). The AUROC measures discrimination, which is the ability to distinguish patients with the event of interest (such as death at a particular time point) from those without. AUROC typically ranges from 0.5 to 1.0, with 0.5 indicating random guessing and 1.0 a perfect prediction, and in general, an AUROC of ≥ 0.7 indicates adequate discrimination for clinical use. Calibration refers to the agreement between the predicted outcomes (in this case, survival probabilities) and the actual outcomes, with a perfect calibration curve having an intercept of 0 and a slope of 1. A positive intercept indicates that the actual survival is generally underestimated by the prediction model, and a negative intercept suggests the opposite (overestimation). When comparing models, an intercept closer to 0 typically indicates better calibration. Calibration can also be summarized as log(O:E), the logarithm scale of the ratio of observed (O) to expected (E) survivors. A log(O:E) > 0 signals an underestimation (the observed survival is greater than the predicted survival); and a log(O:E) < 0 indicates the opposite (the observed survival is lower than the predicted survival). A model with a log(O:E) closer to 0 is generally considered better calibrated. The Brier score is the mean squared difference between the model predictions and the observed outcomes, and it ranges from 0 (best prediction) to 1 (worst prediction). The Brier score captures both discrimination and calibration, and it is considered a measure of overall model performance. In Brier score analysis, the "null model" assigns a predicted probability equal to the prevalence of the outcome and represents a model that adds no new information. A prediction model should achieve a Brier score at least lower than the null-model Brier score to be considered as useful. The DCA was developed as a method to determine whether using a model to inform treatment decisions would do more good than harm. It plots the net benefit of making decisions based on the model's predictions across all possible risk thresholds (or cost-to-benefit ratios) in relation to the two default strategies of treating all or no patients. The care provider can decide on an acceptable risk threshold for the proposed treatment in an individual and assess the corresponding net benefit to determine whether consulting with the model is superior to adopting the default strategies. Finally, we examined whether laboratory data, which were not included in the METSSS model, would have been independently associated with survival after controlling for the METSSS model's predictions by using the multivariable logistic and Cox proportional hazards regression analyses.
Between the two models, only SORG-MLA achieved adequate discrimination (an AUROC of > 0.7) in the entire cohort (of patients treated operatively or with radiation alone) and in the subgroup of patients treated with palliative radiotherapy alone. SORG-MLA outperformed METSSS by a wide margin on discrimination, calibration, and Brier score analyses in not only the entire cohort but also the subgroup of patients whose local treatment consisted of radiotherapy alone. In both the entire cohort and the subgroup, DCA demonstrated that SORG-MLA provided more net benefit compared with the two default strategies (of treating all or no patients) and compared with METSSS when risk thresholds ranged from 0.2 to 0.9 at both 90 days and 1 year, indicating that using SORG-MLA as a decision-making aid was beneficial when a patient's individualized risk threshold for opting for treatment was 0.2 to 0.9. Higher albumin, lower alkaline phosphatase, lower calcium, higher hemoglobin, lower international normalized ratio, higher lymphocytes, lower neutrophils, lower neutrophil-to-lymphocyte ratio, lower platelet-to-lymphocyte ratio, higher sodium, and lower white blood cells were independently associated with better 1-year and overall survival after adjusting for the predictions made by METSSS.
Based on these discoveries, clinicians might choose to consult SORG-MLA instead of METSSS for survival estimation in patients with long-bone metastases presenting for evaluation of local treatment. Basing a treatment decision on the predictions of SORG-MLA could be beneficial when a patient's individualized risk threshold for opting to undergo a particular treatment strategy ranged from 0.2 to 0.9. Future studies might investigate relevant laboratory items when constructing or refining a survival estimation model because these data demonstrated prognostic value independent of the predictions of the METSSS model, and future studies might also seek to keep these models up to date using data from diverse, contemporary patients undergoing both modern operative and nonoperative treatments.
Level III, diagnostic study.
Lee CC
，Chen CW
，Yen HK
，Lin YP
，Lai CY
，Wang JL
，Groot OQ
，Janssen SJ
，Schwab JH
，Hsu FM
，Lin WH
... -
《-》
ResearchGate
(全网免费下载)
钛学术
(全网免费下载)
