Obatoclax potentiates the cytotoxic effect of cytarabine on acute myeloid leukemia cells by enhancing DNA damage

Resistance to cytarabine and anthracycline‐based chemotherapy is a major cause of treatment failure for acute myeloid leukemia (AML) patients. Overexpression of Bcl‐2, Bcl‐xL, and/or Mcl‐1 has been associated with chemoresistance in AML cell lines and with poor clinical outcome of AML patients. Thus, inhibitors of anti‐apoptotic Bcl‐2 family proteins could be novel therapeutic agents. In this study, we investigated how clinically achievable concentrations of obatoclax, a pan‐Bcl‐2 inhibitor, potentiate the antileukemic activity of cytarabine in AML cells. MTT assays in AML cell lines and diagnostic blasts, as well as flow cytometry analyses in AML cell lines revealed synergistic antileukemic activity between cytarabine and obatoclax. Bax activation was detected in the combined, but not the individual, drug treatments. This was accompanied by significantly increased loss of mitochondrial membrane potential. Most importantly, in AML cells treated with the combination, enhanced early induction of DNA double‐strand breaks (DSBs) preceded a decrease of Mcl‐1 levels, nuclear translocation of Bcl‐2, Bcl‐xL, and Mcl‐1, and apoptosis. These results indicate that obatoclax enhances cytarabine‐induced apoptosis by enhancing DNA DSBs. This novel mechanism provides compelling evidence for the clinical use of BH3 mimetics in combination with DNA‐damaging agents in AML and possibly a broader range of malignancies.     

Clonal heterogeneity and rates of specific chromosome gains are risk predictors in childhood high‐hyperdiploid B‐cell acute lymphoblastic leukemia

B‐cell acute lymphoblastic leukemia (B‐ALL) is the commonest childhood cancer. High hyperdiploidy (HHD) identifies the most frequent cytogenetic subgroup in childhood B‐ALL. Although hyperdiploidy represents an important prognostic factor in childhood B‐ALL, the specific chromosome gains with prognostic value in HHD‐B‐ALL remain controversial, and the current knowledge about the hierarchy of chromosome gains, clonal heterogeneity and chromosomal instability in HHD‐B‐ALL remains very limited. We applied automated sequential‐iFISH coupled with single‐cell computational modeling to identify the specific chromosomal gains of the eight typically gained chromosomes in a large cohort of 72 primary diagnostic (DX, n = 62) and matched relapse (REL, n = 10) samples from HHD‐B‐ALL patients with either favorable or unfavorable clinical outcome in order to characterize the clonal heterogeneity, specific chromosome gains and clonal evolution. Our data show a high degree of clonal heterogeneity and a hierarchical order of chromosome gains in DX samples of HHD‐B‐ALL. The rates of specific chromosome gains and clonal heterogeneity found in DX samples differ between HHD‐B‐ALL patients with favorable or unfavorable clinical outcome. In fact, our comprehensive analyses at DX using a computationally defined risk predictor revealed low levels of trisomies +18+10 and low levels of clonal heterogeneity as robust relapse risk factors in minimal residual disease (MRD)‐negative childhood HHD‐B‐ALL patients: relapse‐free survival beyond 5 years: 22.1% versus 87.9%, P < 0.0001 and 33.3% versus 80%, P < 0.0001, respectively. Moreover, longitudinal analysis of matched DX‐REL HHD‐B‐ALL samples revealed distinct patterns of clonal evolution at relapse. Our study offers a reliable prognostic sub‐stratification of pediatric MRD‐negative HHD‐B‐ALL patients.     

Clinical significance of IDC‐P as predictive factor after intensity‐modulated radiation therapy

The clinical significance of intraductal carcinoma of the prostate (IDC‐P) in men with nonmetastatic prostate cancer (PCa) treated with high‐dose external‐beam radiation therapy remains unclear. The aim of this study was to evaluate the impact of IDC‐P in men who received intensity‐modulated radiation therapy (IMRT) for nonmetastatic PCa. All patients with high‐risk (H‐R) and very high–risk (VH‐R) PCa who received IMRT between September 2000 and December 2013 at our institution were analyzed retrospectively. We re‐reviewed biopsy cores for the presence of IDC‐P. Treatment consisted of IMRT (median: 78 Gy at 2 Gy per fraction) plus 6‐month neoadjuvant hormonal therapy (HT). In total, 154 consecutive patients with H‐R and VH‐R PCa were analyzed. Intraductal carcinoma of the prostate was present in 27.9% (n = 43). The median follow‐up period was 8.4 years. The 10‐year PCa‐specific survival, biochemical failure (BF), clinical failure, and castration‐resistant PCa rates were 90.0%, 47.8%, 27.5%, and 24.5% in patients with IDC‐P, and 96.6%, 32.6%, 10.8%, and 7.0% in those without IDC‐P, respectively (p = 0.12, 0.04, 0.0031, and 0.012, respectively). In multivariable analysis, IDC‐P was not identified as an independent predictive factor for BF (p = 0.26). The presence of IDC‐P was correlated with a significantly higher incidence of disease progression in men with H‐R and VH‐R PCa who received IMRT, although it was not identified as an independent predictive factor for BF. Further investigations are needed to determine the significance of IDC‐P as an independent predictive factor for survival outcomes.     

Identification of Very Low‐Risk Subgroups of Patients with Primary Mediastinal Large B‐Cell Lymphoma Treated with R‐CHOP

BackgroundR‐CHOP can cure approximately 75% of patients with primary mediastinal large B‐cell lymphoma (PMLBCL), but prognostic factors have not been sufficiently evaluated yet. R‐da‐ EPOCH is potentially more effective but also more toxic than R‐CHOP. Reliable prognostic classification is needed to guide treatment decisions.Materials and MethodsWe analyzed the impact of clinical prognostic factors on the outcome of 332 PMLBCL patients ≤65 years treated with R‐CHOP ± radiotherapy in a multicenter setting in Greece and Cyprus.ResultsWith a median follow‐up of 69 months, 5‐year freedom from progression (FFP) was 78% and 5‐year lymphoma specific survival (LSS) was 89%. On multivariate analysis, extranodal involvement (E/IV) and lactate dehydrogenase (LDH) ≥2 times upper limit of normal (model A) were significantly associated with FFP; E/IV and bulky disease (model B) were associated with LSS. Both models performed better than the International Prognostic Index (IPI) and the age‐adjusted IPI by Harrel''s C rank parameter and Akaike information criterion. Both models A and B defined high‐risk subgroups (13%–27% of patients [pts]) with approximately 19%–23% lymphoma‐related mortality. They also defined subgroups composing approximately one‐fourth or one‐half of the patients, with 11% risk of failure and only 1% or 4% 5‐year lymphoma‐related mortality.ConclusionThe combination of E/IV with either bulky disease or LDH ≥2 times upper limit of normal defined high‐risk but not very‐high‐risk subgroups. More importantly, their absence defined subgroups comprising approximately one‐fourth or one‐half of the pts, with 11% risk of failure and minimal lymphoma‐related mortality, who may not need more intensive treatment such as R‐da‐EPOCH.Implications for PracticeBy analyzing the impact of baseline clinical characteristics on outcomes of a large cohort of patients with primary mediastinal large B‐cell lymphoma homogeneously treated with R‐CHOP with or without radiotherapy, we developed novel prognostic indices which can aid in deciding which patients can be adequately treated with R‐CHOP and do not need more intensive regimens such as R‐da‐EPOCH. The new indices consist of objectively determined characteristics (extranodal disease or stage IV, bulky disease, and markedly elevated serum lactate dehydrogenase), which are readily available from standard initial staging procedures and offer better discrimination compared with established risk scores (International Prognostic Index [IPI] and age‐adjusted IPI).     

E‐cadherin is a robust prognostic biomarker in colorectal cancer and low expression is associated with sensitivity to inhibitors of topoisomerase,aurora, and HSP90 in preclinical models

Cell–cell and cell–matrix adhesion proteins that have been implicated in colorectal epithelial integrity and epithelial‐to‐mesenchymal transition could be robust prognostic and potential predictive biomarkers for standard and novel therapies. We analyzed in situ protein expression of E‐cadherin (ECAD), integrin β4 (ITGB4), zonula occludens 1 (ZO‐1), and cytokeratins in a single‐hospital series of Norwegian patients with colorectal cancer (CRC) stages I–IV (n = 922) using multiplex fluorescence‐based immunohistochemistry (mfIHC) on tissue microarrays. Pharmacoproteomic associations were explored in 35 CRC cell lines annotated with drug sensitivity data on > 400 approved and investigational drugs. ECAD, ITGB4, and ZO‐1 were positively associated with survival, while cytokeratins were negatively associated with survival. Only ECAD showed independent prognostic value in multivariable Cox models. Clinical and molecular associations for ECAD were technically validated on a different mfIHC platform, and the prognostic value was validated in another Norwegian series (n = 798). In preclinical models, low and high ECAD expression differentially associated with sensitivity to topoisomerase, aurora, and HSP90 inhibitors, and EGFR inhibitors. E‐cadherin protein expression is a robust prognostic biomarker with potential clinical utility in CRC.     

Merkel cell polyomavirus–negative Merkel cell carcinoma is associated with JAK‐STAT and MEK‐ERK pathway activation

Merkel cell polyomavirus (MCPyV) is monoclonally integrated into the genomes of approximately 80% of Merkel cell carcinomas (MCCs). While the presence of MCPyV affects the clinicopathological features of MCC, the molecular mechanisms of MCC pathogenesis after MCPyV infection are unclear. This study investigates the association between MCPyV infection and activation of the MEK‐ERK and JAK‐STAT signaling pathways in MCC to identify new molecular targets for MCC treatment. The clinicopathological characteristics of 30 MCPyV‐positive and 20 MCPyV‐negative MCC cases were analyzed. The phosphorylation status of MEK, ERK, JAK, and STAT was determined by immunohistochemical analysis. The activation status of the MEK‐ERK and JAK‐STAT pathways and the effects of a JAK inhibitor (ruxolitinib) was analyzed in MCC cell lines. Immunohistochemically, the expression of pJAK2 (P = .038) and pERK1/2 (P = .019) was significantly higher in MCPyV‐negative than in MCPyV‐positive MCCs. Male gender (hazard ratio [HR] 2.882, P = .039), older age (HR 1.137, P < .001), negative MCPyV status (HR 0.324, P = .013), and advanced cancer stage (HR 2.672, P = .041) were identified as unfavorable prognostic factors; however, the phosphorylation states of JAK2, STAT3, MEK1/2, and ERK1/2 were unrelated to the prognosis. The inhibition of cell proliferation by ruxolitinib was greater in MCPyV‐negative MCC cell lines than in an MCPyV‐positive MCC cell line. The expression of pERK1/2 and pMEK was higher in MCPyV‐negative than in MCPyV‐positive cell lines. These results suggest that activation of the JAK2 and MEK‐ERK pathways was more prevalent in MCPyV‐negative than in MCPyV‐positive MCC and the JAK inhibitor ruxolitinib inhibited MEK‐ERK pathway activation. Consequently, the JAK‐STAT and MEK‐ERK signaling pathways may be potential targets for MCPyV‐negative MCC treatment.     

miR‐200a/b/‐429 downregulation is a candidate biomarker of tumor radioresistance and independent of hypoxia in locally advanced cervical cancer

Many patients with locally advanced cervical cancer experience recurrence within the radiation field after chemoradiotherapy. Biomarkers of tumor radioresistance are required to identify patients in need of intensified treatment. Here, the biomarker potential of miR‐200 family members was investigated in this disease. Also, involvement of tumor hypoxia in the radioresistance mechanism was determined, using a previously defined 6‐gene hypoxia classifier. miR‐200 expression was measured in pretreatment tumor biopsies of an explorative cohort (n = 90) and validation cohort 1 (n = 110) by RNA sequencing. Publicly available miR‐200 data of 79 patients were included for the validation of prognostic significance. A score based on expression of the miR‐200a/b/‐429 (miR‐200a, miR‐200b, and miR‐429) cluster showed prognostic significance in all cohorts. The score was significant in multivariate analysis of central pelvic recurrence. No association with distant recurrence or hypoxia status was found. Potential miRNA target genes were identified from gene expression profiles and showed enrichment of genes in extracellular matrix organization and cell adhesion. miR‐200a/b/‐429 overexpression had a pronounced radiosensitizing effect in tumor xenografts, whereas the effect was minor in vitro. In conclusion, miR‐200a/b/‐429 downregulation is a candidate biomarker of central pelvic recurrence and seems to predict cell adhesion‐mediated tumor radioresistance independent of clinical markers and hypoxia.     

A novel anti‐c‐Kit antibody–drug conjugate to treat wild‐type and activating‐mutant c‐Kit‐positive tumors

c‐Kit overexpression and activating mutations, which are reported in various cancers, including gastrointestinal stromal tumor (GIST), small‐cell lung cancer (SCLC), acute myeloid leukemia, acral melanoma, and systemic mastocytosis (SM), confer resistance to tyrosine kinase inhibitors (TKIs). To overcome TKI resistance, an anti‐c‐Kit antibody–drug conjugate was developed in this study to treat wild‐type and mutant c‐Kit‐positive cancers. NN2101, a fully human IgG1, was conjugated to DM1, a microtubule inhibitor, through N‐succinimidyl‐4‐(N‐maleimidomethyl) cyclohexane‐1‐carboxylate (SMCC) (to give NN2101‐DM1). The antitumor activity of NN2101‐DM1 was evaluated in vitro and in vivo using various cancer cell lines. NN2101‐DM1 exhibited potent growth‐inhibitory activities against c‐Kit‐positive cancer cell lines. In a mouse xenograft model, NN2101‐DM1 exhibited potent growth‐inhibitory activities against imatinib‐resistant GIST and SM cells. In addition, NN2101‐DM1 exhibited a significantly higher anti‐cancer effect than carboplatin/etoposide against SCLC cells where c‐Kit does not mediate cancer pathogenesis. Furthermore, the combination of NN2101‐DM1 with imatinib in imatinib‐sensitive GIST cells induced complete remission compared with treatment with NN2101‐DM1 or imatinib alone in mouse xenograft models. These results suggest that NN2101‐DM1 is a potential therapeutic agent for wild‐type and mutant c‐Kit‐positive cancers.     

Suppression of multiple anti‐apoptotic BCL2 family proteins recapitulates the effects of JAK2 inhibitors in JAK2V617F driven myeloproliferative neoplasms

Several lines of research suggest that Bcl‐xL‐mediated anti‐apoptotic effects may contribute to the pathogenesis of myeloproliferative neoplasms driven by JAK2V617F and serve as therapeutic target. Here, we used a knock‐in JAK2V617F mouse model and confirmed that Bcl‐xL was overexpressed in erythroid progenitors. The myeloproliferative neoplasm (MPN)‐induced phenotype in the peripheral blood by conditional knock‐in of JAK2V617F was abrogated by conditional knockout of Bcl2l1, which presented anemia and thrombocytopenia independently of JAK2 mutation status. Mx1‐Cre Jak2V617^W/VF/Bcl2l1^f/f mice presented persistent splenomegaly as a result of extramedullary hematopoiesis and pro‐apoptotic stimuli in terminally differentiated erythroid progenitors. The pan‐BH3 mimetic inhibitor obatoclax showed superior cytotoxicity in JAK2V617F cell models, and reduced clonogenic capacity in ex vivo assay using Vav‐Cre Jak2V617F bone marrow cells. Both ruxolitinib and obatoclax significantly reduced spleen weights in a murine Jak2V617F MPN model but did not show additive effect. The tumor burden reduction was observed with either ruxolitinib or obatoclax in terminal differentiation stage neoplastic cells but not in myeloid‐erythroid precursors. Therefore, disrupting the BCL2 balance is not sufficient to treat MPN at the stem cell level, but it is certainly an additional option for controlling the critical myeloid expansion of the disease.     

Cost‐effectiveness of precision diagnostic testing for precision medicine approaches against non‐small‐cell lung cancer: A systematic review

Precision diagnostic testing (PDT) employs appropriate biomarkers to identify cancer patients that may optimally respond to precision medicine (PM) approaches, such as treatments with targeted agents and immuno‐oncology drugs. To date, there are no published systematic appraisals evaluating the cost‐effectiveness of PDT in non‐small‐cell lung cancer (NSCLC). To address this gap, we conducted Preferred Reporting Items for Systematic Reviews and Meta‐Analyses searches for the years 2009–2019. Consolidated Health Economic Evaluation Reporting Standards were employed to screen, assess and extract data. Employing base costs, life years gained or quality‐adjusted life years, as well as willingness‐to‐pay (WTP) threshold for each country, net monetary benefit was calculated to determine cost‐effectiveness of each intervention. Thirty‐seven studies (50%) were included for analysis; a further 37 (50%) were excluded, having failed population‐, intervention‐, comparator‐, outcomes‐ and study‐design criteria. Within the 37 studies included, we defined 64 scenarios. Eleven scenarios compared PDT‐guided PM with non‐guided therapy [epidermal growth factor receptor (EGFR), n = 5; programmed death‐ligand 1 (PD‐L1), n = 6]. Twenty‐eight scenarios compared PDT‐guided PM with chemotherapy alone (anaplastic lymphoma kinase, n = 3; EGFR, n = 17; PD‐L1, n = 8). Twenty‐five scenarios compared PDT‐guided PM with chemotherapy alone, while varying the PDT approach. Thirty‐four scenarios (53%) were cost‐effective, 28 (44%) were not cost‐effective, and two were marginal, dependent on their country’s WTP threshold. When PDT‐guided therapy was compared with a therapy‐for‐all patients approach, all scenarios (100%) proved cost‐effective. Seven of 37 studies had been structured appropriately to assess PDT‐PM cost‐effectiveness. Within these seven studies, all evaluated scenarios were cost‐effective. However, 81% of studies had been poorly designed. Our systematic analysis implies that more robust health economic evaluation could help identify additional approaches towards PDT cost‐effectiveness, underpinning value‐based care and enhanced outcomes for patients with NSCLC.     

Pre‐ and post‐treatment blood‐based genomic landscape of patients with ROS1 or NTRK fusion‐positive solid tumours treated with entrectinib

Genomic tumour profiling informs targeted treatment options. Entrectinib is a tyrosine kinase inhibitor with efficacy in NTRK fusion‐positive (‐fp) solid tumours and ROS1‐fp non‐small cell lung cancer. FoundationOne® Liquid CDx (F1L CDx), a non‐invasive in  vitro next‐generation sequencing (NGS)‐based diagnostic, detects genomic alterations in plasma circulating tumour DNA (ctDNA). We evaluated the clinical validity of F1L CDx as an aid in identifying patients with NTRK‐fp or ROS1‐fp tumours and assessed the genomic landscape pre‐ and post‐entrectinib treatment. Among evaluable pre‐treatment clinical samples (N = 85), positive percentage agreements between F1L CDx and clinical trial assays (CTAs) were 47.4% (NTRK fusions) and 64.5% (ROS1 fusions); positive predictive value was 100% for both. The objective response rate for CTA⁺ F1L CDx⁺ patients was 72.2% in both cohorts. The median duration of response significantly differed between F1L CDx⁺ and F1L CDx⁻ samples in ROS1‐fp (5.6 vs. 17.3 months) but not NTRK‐fp (9.2 vs. 12.9 months) patients. Fifteen acquired resistance mutations were detected. We conclude that F1L CDx is a clinically valid complement to tissue‐based testing to identify patients who may benefit from entrectinib and those with acquired resistance mutations associated with disease progression.     

High PTX3 expression is associated with a poor prognosis in diffuse large B‐cell lymphoma

Tumor‐associated macrophages (TAMs) are associated with a poor prognosis of diffuse large B‐cell lymphoma (DLBCL). As macrophages are heterogeneous, the immune polarization and their pathological role warrant further study. We characterized the microenvironment of DLBCL by immunohistochemistry in a training set of 132 cases, which included 10 Epstein–Barr virus‐encoded small RNA (EBER)‐positive and five high‐grade B‐cell lymphomas, with gene expression profiling in a representative subset of 37 cases. Diffuse large B‐cell lymphoma had a differential infiltration of TAMs. The high infiltration of CD68 (pan‐macrophages), CD16 (M1‐like), CD163, pentraxin 3 (PTX3), and interleukin (IL)‐10‐positive macrophages (M2c‐like) and low infiltration of FOXP3‐positive regulatory T lymphocytes (Tregs) correlated with poor survival. Activated B cell‐like DLBCL was associated with high CD16, CD163, PTX3, and IL‐10, and EBER‐positive DLBCL with high CD163 and PTX3. Programmed cell death‐ligand 1 positively correlated with CD16, CD163, IL‐10, and RGS1. In a multivariate analysis of overall survival, PTX3 and International Prognostic Index were identified as the most relevant variables. The gene expression analysis showed upregulation of genes involved in innate and adaptive immune responses and macrophage and Toll‐like receptor pathways in high PTX3 cases. The prognostic relevance of PTX3 was confirmed in a validation set of 159 cases. Finally, in a series from Europe and North America ({"type":"entrez-geo","attrs":{"text":"GSE10846","term_id":"10846"}}GSE10846, R‐CHOP‐like treatment, n = 233) high gene expression of PTX3 correlated with poor survival, and moderately with CSF1R, CD16, MITF, CD163, MYC, and RGS1. Therefore, the high infiltration of M2c‐like immune regulatory macrophages and low infiltration of FOXP3‐positive Tregs is associated with a poor prognosis in DLBCL, for which PTX3 is a new prognostic biomarker.     

Tetraspanin 1 promotes endometriosis leading to ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) reportedly develops from endometriosis. However, the molecular mechanism underlying its malignant progression to OCCC remains elusive. This study aimed to identify an essential gene in the malignant transformation of endometriosis to OCCC. We performed RNA sequencing in formalin‐fixed, paraffin‐embedded (FFPE) tissues of endometriosis (n = 9), atypical endometriosis (AtyEm) (n = 18), adjacent endometriosis to OCCC (AdjEm) (n = 7), and OCCC (n = 17). We found that tetraspanin 1 (TSPAN1) mRNA level was significantly increased by 2.4‐ (DESeq2) and 3.4‐fold (edgeR) in AtyEm and by 80.7‐ (DESeq2) and 101‐fold (edgeR) in OCCC relative to endometriosis. We confirmed that TSPAN1 protein level was similarly overexpressed in OCCC tissues and cell lines. In immortalized endometriosis cell lines, TSPAN1 overexpression enhanced cell growth and invasion. Mechanistically, TSPAN1 triggered AMP‐activated protein kinase (AMPK) activity, promoting endometriosis and cell growth. Upregulated levels of TSPAN1 are considered an early event in the development of high‐risk endometriosis that could progress to ovarian cancer. Our study suggests the potential of TSPAN1 as a screening candidate for high‐risk endometriosis.     

Homeopathic Treatment as an Add‐On Therapy May Improve Quality of Life and Prolong Survival in Patients with Non‐Small Cell Lung Cancer: A Prospective,Randomized, Placebo‐Controlled,Double‐Blind,Three‐Arm,Multicenter Study

Lessons Learned

• Conventional medicine and homeopathy work well together.
• Quality of life improves with additive homeopathy in patients with non‐small cell lung cancer (NSCLC).
• Survival improves with additive homeopathy in patients with NSCLC.

BackgroundPatients with advanced non‐small cell lung cancer (NSCLC) have limited treatment options. Alongside conventional anticancer treatment, additive homeopathy might help to alleviate side effects of conventional therapy. The aim of the present study was to investigate whether additive homeopathy might influence quality of life (QoL) and survival in patients with NSCLC.MethodsIn this prospective, randomized, placebo‐controlled, double‐blind, three‐arm, multicenter, phase III study, we evaluated the possible effects of additive homeopathic treatment compared with placebo in patients with stage IV NSCLC, with respect to QoL in the two randomized groups and survival time in all three groups. Treated patients visited the outpatients'' centers every 9 weeks: 150 patients with stage IV NSCLC were included in the study; 98 received either individualized homeopathic remedies (n = 51) or placebo (n = 47) in a double‐blinded fashion; and 52 control patients without any homeopathic treatment were observed for survival only. The constituents of the different homeopathic remedies were mainly of plant, mineral, or animal origin. The remedies were manufactured by stepwise dilution and succussion, thereby preparing stable Good Manufacturing Practice grade formulations.ResultsQoL as well as functional and symptom scales showed significant improvement in the homeopathy group when compared with placebo after 9 and 18 weeks of homeopathic treatment (p < .001). Median survival time was significantly longer in the homeopathy group (435 days) versus placebo (257 days; p = .010) as well as versus control (228 days; p < .001). Survival rate in the homeopathy group differed significantly from placebo (p = .020) and from control (p < .001).ConclusionQoL improved significantly in the homeopathy group compared with placebo. In addition, survival was significantly longer in the homeopathy group versus placebo and control. A higher QoL might have contributed to the prolonged survival. The study suggests that homeopathy positively influences not only QoL but also survival. Further studies including other tumor entities are warranted.     

Comprehensive cross‐platform comparison of methods for non‐invasive EGFR mutation testing: results of the RING observational trial

Several platforms for noninvasive EGFR testing are currently used in the clinical setting with sensitivities ranging from 30% to 100%. Prospective studies evaluating agreement and sources for discordant results remain lacking. Herein, seven methodologies including two next‐generation sequencing (NGS)‐based methods, three high‐sensitivity PCR‐based platforms, and two FDA‐approved methods were compared using 72 plasma samples, from EGFR‐mutant non‐small‐cell lung cancer (NSCLC) patients progressing on a first‐line tyrosine kinase inhibitor (TKI). NGS platforms as well as high‐sensitivity PCR‐based methodologies showed excellent agreement for EGFR‐sensitizing mutations (K = 0.80–0.89) and substantial agreement for T790M testing (K = 0.77 and 0.68, respectively). Mutant allele frequencies (MAFs) obtained by different quantitative methods showed an excellent reproducibility (intraclass correlation coefficients 0.86–0.98). Among other technical factors, discordant calls mostly occurred at mutant allele frequencies (MAFs) ≤ 0.5%. Agreement significantly improved when discarding samples with MAF ≤ 0.5%. EGFR mutations were detected at significantly lower MAFs in patients with brain metastases, suggesting that these patients risk for a false‐positive result. Our results support the use of liquid biopsies for noninvasive EGFR testing and highlight the need to systematically report MAFs.

Abbreviations

BEAMing

beads, emulsion, amplification, and magnetics

cfDNA

circulating free DNA, cell‐free DNA

cobas

cobas^® EGFR Mutation Test v2 (Roche Diagnostics)

ctDNA

circulating tumor DNA

CUSUM

cumulative sum

ddPCR

droplet digital polymerase chain reaction

dPCR

digital polymerase chain reaction

EGFR

epidermal growth factor receptor

FFPE

formalin‐fixed, paraffin‐embedded

ICC

intraclass correlation coefficient

MAF

mutant allele frequency

NGS platforms

Ion S5™ XL and GeneRead™

NGS

next‐generation sequencing

NSCLC

non‐small‐cell lung cancer

PNA‐Q‐PCR

peptic nucleic acid probe‐based real‐time polymerase chain reaction

Therascreen

Therascreen EGFR Plasma RGQ PCR Kit (QIAgen)

TKI

tyrosine kinase inhibitor