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Los pacientes con CPNM metastásico portadores de una fusión del gen ROS1 suelen experimentar un control prolongado de la enfermedad con inhibidores de la tirosina quinasa (TKI) dirigidos a ROS1, aunque hay una heterogeneidad clínica significativa, debida en parte a la presencia de alteraciones genómicas concurrentes. Presentamos el caso de un paciente con CPNM metastásico, con fusión de ROS1 acompañada de una mutación en KRAS p.G12C detectada durante el diagnóstico, quien mantuvo la enfermedad bajo control durante un breve periodo con entrectinib, un TKI de ROS1. Cuando se observó progresión, el paciente continuó con entrectinib y comenzó a recibir sotorasib, un inhibidor de KRAS p.G12C de bajo peso molecular. Una línea celular derivada del paciente generada durante la progresión mientras recibía entrectinib demostró una mejor capacidad de respuesta a los TKI cuando se trató con entrectinib y sotorasib. La dependencia del crecimiento de la línea celular tanto con respecto a ROS1 como a KRAS p.G12C se reflejó además en las distintas vías de señalización corriente abajo activadas por cada impulsor. No se observó beneficio clínico con la terapia combinada de entrectinib y sotorasib, posiblemente debido a una amplificación evolutiva de KRAS p.G12C identificada en pruebas moleculares repetidas. Este caso respalda la necesidad de establecer perfiles moleculares amplios en pacientes con CPNM metastásico para disponer de información con posible valor terapéutico y pronóstico.

Background: Although monosomal karyotype (MK) has been recognized as an adverse risk factor in adult acute myeloid leukemia (AML), its impact on pediatric AML outcomes remains unclear. Methods: Clinical characteristics, cytogenetic and molecular profiling, and prognostic impact of MK were analyzed in a cohort of 284 children with AML in a Chinese population. Results: MK was present in 14 (4.5%) cases of the study cohort. MK-AML showed low prevalence of TP53 mutant and 17p13 aberrations. Children with MK-AML had shorter overall survival (OS) and event-free survival (EFS) compared to those with intermediate-risk features (OS: 26 ± 9.7% vs. 2 ± 1.5%, p

Objective To investigate O‐6‐methylguanine‐DNA methyltransferase (MGMT) gene promoter methylation in humoral tissue as biomarker for lung cancer diagnosis by pooling relevant open published data. Methods Clinical studies relevant to MGMT gene promoter methylation and lung cancer were systematic electronic searched in the databases of Medline, EMBASE, Ovid, Web of Science, and CNKI. Data of true positive (tp), false positive (fp), false negative (fn), and true negative (tn) were extracted from the included studies and made combination. The diagnostic sensitivity, specificity, diagnostic odds ratio (DOR) and summary receiver operating characteristic (SROC) of MGMT gene methylation for lung cancer diagnosis were pooled. Results Twelve studies were included in the meta‐analysis. The diagnostic sensitivity, specificity, DOR were 0.39 (95% CI = 0.31–0.49) 0.92 (95% CI = 0.77–0.97), and 4.20 (95% CI = 2.09–8.44), respectively under random effect model. The SROC of MGMT gene methylation for lung cancer diagnosis was 0.58 (95% CI = 0.53–0.62). Conclusion MGMT methylation rate was higher in plasma and bronchoalveolar lavage fluid (BLAF) of lung cancer cases compared to controls. High diagnostic specificity indicated that MGMT methylation in plasma and BLAF can be applied as lung cancer confirmation test., Because of significant statistical heterogeneity across the included 12 studies, the data was pooled in random effect model. The diagnostic sensitivity, specificity 0.39 (95% CI = 0.31–0.49) and 0.92 (95% CI = 0.77–0.97), respectively under random effect model.

Chromosomal microarray (CMA) is now widely used as first-tier testing for the detection of copy number variants (CNVs) and absence of heterozygosity (AOH) in patients with multiple congenital anomalies (MCA), autism spectrum disorder (ASD), developmental delay (DD), and/or intellectual disability (ID). Chromosome analysis is commonly used to complement CMA in the detection of balanced genomic aberrations. However, the cost-effectiveness and the impact on clinical management of chromosome analysis concomitant with CMA were not well studied, and there is no consensus on how to best utilize these two tests. To assess the clinical utility and cost-effectiveness of chromosome analysis concomitant with CMA in patients with MCA, ASD, DD, and/or ID, we retrospectively analyzed 3,360 postnatal cases for which CMA and concomitant chromosome analysis were performed in the Colorado Genetic Laboratory (CGL) at the University Of Colorado School Of Medicine. Chromosome analysis alone yielded a genetic diagnosis in two patients (0.06%) and contributed additional information to CMA results in 199 (5.92%) cases. The impact of abnormal chromosome results on patient management was primarily related to counseling for reproductive and recurrence risks assessment (101 cases, 3.01%) while a few (5 cases, 0.15%) led to changes in laboratory testing and specialist referral (25 cases, 0.74%). The incremental cost-effectiveness ratio (ICER) of combined testing demonstrated the cost of each informative chromosome finding was significantly higher for patients with clinically insignificant (CI) CMA findings versus clinically significant (CS) CMA results. Our results suggest that a stepwise approach with CMA testing with reflex to chromosome analysis on cases with CS CMA findings is a more cost-effective testing algorithm for patients with MCA, ASD, and/or DD/ID.

Background: 21q22 amplification is a rare cytogenetic aberration in acute myeloid leukemia (AML). So far, the cytogenomic and molecular features and clinical correlation of 21q22 amplification in AML have not been well-characterized. Case Presentation: Here, we describe a case series of three AML patients with amplified 21q22 identified by fluorescence in situ hybridization (FISH) using a RUNX1 probe. Two of these patients presented with therapy-related AML (t-AML) secondary to chemotherapy, while the third had de novo AML. There was one case each of FAB M0, M1 and M4. Morphologic evidence of dysplasia was identified in both t-AML cases. Phenotypic abnormalities of the myeloblasts were frequently observed. Extra copies of 21q22 were present on chromosome 21 and at least one other chromosome in two cases. Two showed a highly complex karyotype. Microarray analysis of 21q22 amplification in one case demonstrated alternating levels of high copy number gain split within the RUNX1 locus at 21q22, a pattern distinct from the iAMP21 profile reported in B-cell precursor acute lymphoblastic leukemia (B-ALL). The same patient also had mutated TP53. Two patients died at 1.5 and 11 months post-treatment, while the third elected palliative care and died within 2 weeks. Conclusions: Our results provide further evidence that 21q22 amplification in AML is associated with complex karyotypes, TP53 aberrations, and poor outcomes. Furthermore, we demonstrate that 21q22 amplification is not always intrachromosomally localized to chromosome 21 and could be a result of structural aberrations involving 21q22 and other chromosomes.

Chromosome microarray analysis (CMA) has become the first-tier testing for chromosomal abnormalities and copy number variations (CNV). This review described the clinical validation of CMA, the development and updating of technical standards and guidelines and their diagnostic impacts. The main focuses were on the development and updating of expert consensus, practice resources, and a series of technical standards and guidelines through systematic review of case series with CMA application in the literature. Expert consensus and practice resource supported the use of CMA as the first-tier testing for detecting chromosomal abnormalities and CNV in developmental and intellectual disabilities, multiple congenital anomalies and autism. The standards and guidelines have been applied to pre- and postnatal testing for constitutional CNV and tumor testing for acquired CNV. CMA has significantly improved the diagnostic yields but still needs to overcome its technical limitations and face challenges of new technologies. Guiding and governing CMA through expert consensus, practice resource, standards and guidelines in the United States has provided effective and safe diagnostic services to patients and their families, reliable diagnosis on related genetic diseases for clinical database and basic research, and references for clinical translation of new technologies.

Multiple primary malignant tumors (MPMTs) are rarely seen among the patients with malignant neoplasms. Moreover, the existence of five MPMTs in the same patient is an extremely rare phenomenon. In this case, a 42‐year‐old male patient developed five metachronous MPMTs within 16 years and the duration between each malignant tumor shortened with the progression of the disease. Multidisciplinary treatments were used on this patient and he fought against the cancers until the end of his life. Our report provides us with a new awareness of MPMTs, which should be considered when we come across with cancer patients who develop various unexplainable symptoms after the diagnosis of the first neoplasm.

Background: GATA2 is important for the regulation of development and proliferation of early pluripotent hematopoietic precursors, and precise GATA2 expression is critical in myeloid lineage maturation. GATA2 mutations have been reported in acute myeloid leukemia (AML) but only few studies of GATA2 mutations and overexpression in pediatric AML have been reported so far and none was in Chinese populations. Method: We analyzed GATA mutation status and expression state in a cohort of 309 Chinese children with AML. We also assessed the impact of GATA2 mutations and overexpression on outcomes in our pediatric AML cohort. Results: GATA2 mutations were observed in 2.6% of the patients and they were clustered in the ZF1 and ZF2 domains. GATA2 overexpression was detected in 72.7% of the cases, with the highest and lowest expression levels observed in adverse subgroup and favorable subgroup, respectively. In the entire cohort, shorter overall survival (OS) and event-free survival (EFS) showed a stronger correlation with GATA2 overexpression than with normal GATA2 levels (OS: p = 0.021; EFS: p = 0.018). Such an inverse relationship between GATA2 overexpression and survivals was evident among AML patients with normal cytogenetics (OS: p = 0.037; EFS: p = 0.024) and adverse features (OS: p = 0.021; EFS: p = 0.023), but not observed in favorable subgroup (OS: p = 0.323; EFS: p = 0.342), suggesting that the negative influence of GATA2 overexpression on pediatric AML outcomes is risk subgroup specific. Conclusion: Our results demonstrate that GATA2 mutations in pediatric AML are rare, and are primarily clustered in the zinc finger (ZF) domains. Our results suggest that GATA2 overexpression exerts a negative influence on survivals, and the effect is risk subgroup specific in pediatric childhood AML. Keywords: Acute myeloid leukemia, Pediatric, GATA2, Overexpression, Outcome

ALK/ROS1 TKIs have significantly improved outcomes for patients with ALK/ROS1+ lung adenocarcinoma. However, drug resistance inevitably develops, leading to disease progression. Approximately 1/3 of patients resistant to ROS1 TKIs demonstrate on-target ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in most patients remains unknown/poorly characterized. To model and characterize acquired resistance to crizotinib, we used ROS1+ and ALK+ primary, patient tumor-derived non-small cell lung cancer (NSCLC) cell lines to generate acquired resistance models to the ALK/ROS1 inhibitor crizotinib. We used several techniques to probe the mechanisms driving resistance: DNA and RNA sequencing (seq), fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. The crizotinib-resistant line initially driven by a TPM3-ROS1 fusion (CUTO28-CR) displayed exquisite sensitivity to Src family kinase (SFK) TKIs in proliferation assays, which was modestly improved by addition of ERBB TKI afatinib. Bulk RNA seq revealed upregulation of SFKs Fyn and Src transcripts (141- and 2.31-fold, respectively). Interphase FISH revealed modest EGFR gene amplification in CUTO28-CR cells vs parental (EGFR:CEP7 ratio 2.1 vs 1.0), and RNA seq revealed upregulation of EGFR and HER2 transcripts (1.79- and 2.45-fold, respectively). Western blot analysis confirmed upregulation of Fyn at the protein level. CUTO28-CR cells treated with a SFK TKI revealed dose-dependent deactivation of Akt signaling, and addition of afatinib caused inhibition of MAPK signaling. Stimulation with epidermal growth factor (EGF) enhanced MAPK signaling in CUTO28-CR cells more than in parental cells, consistent with EGFR/HER2 dependence. Because of a noted propensity for the EML4-ALK line (H3122) to upregulate ERBB kinase activity under sustained ALK TKI treatment, we maintained these cells in low dose (25nM) afatinib while driving crizotinib resistance to identify additional bypass signaling pathways. These H3122-CAR cells were partially resensitized to crizotinib with the addition of SFK TKIs, and the addition of afatinib with SFK TKIs further resensitized these resistant cells to crizotinib. Bulk RNA seq of H3122-CAR cells revealed upregulation of Fyn and Src (2.64- and 3.63-fold, respectively), and western blot analysis confirmed upregulation at the protein level. In conclusion, we demonstrated increased SFK (particularly Fyn and/or Src) expression and/or activation is a mechanism utilized to drive bypass signaling, in cooperation with ERBB family kinases, to circumvent treatment with ALK/ROS1 TKI crizotinib. To our knowledge, this is the first report of SFK-driven bypass signaling as a mechanism of acquired resistance to an ALK/ROS1 TKI in NSCLC. The upregulation of Src and/or Fyn in NSCLC biopsies may guide the exploration of combination SFK TKIs with ALK/ROS1 TKIs. Citation Format: Logan C. Tyler, Nan Chen, Anh T. Le, Hala Nijmeh, Liming Bao, Robert C. Doebele. Fyn and Src cooperate with ERBB family to drive resistance to crizotinib in ROS1+ and ALK+ NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1102.

Patients with metastatic non-small cell lung cancer (NSCLC) positive for a ROS proto-oncogene 1 (ROS1) fusion usually experience significant disease response and long-term control while on treatment with a ROS1 targeting tyrosine kinase inhibitor (TKI) but significant clinical heterogeneity exists. The presence of co-occurring genomic alterations likely contribute to this diversity in clinical outcomes. Here, we report on a patient with metastatic NSCLC with a ROS1 fusion and Kirsten rat sarcoma viral oncogene homolog (KRAS) G12C mutation detected at diagnosis who experienced a short duration of disease control on targeted therapy. The patient was initially treated with entrectinib, a ROS1 TKI, and, at progression, also received sotorasib, a small molecule inhibitor of KRAS G12C, in combination. Using specimens from therapeutically relevant time points including at diagnosis, at time of resistance to entrectinib, and at time of resistance to entrectinib and concurrent sotorasib, we performed broad next generation sequencing and FISH studies to detail the development of therapy resistance. Variant allele frequency of KRAS G12C was increased at each time point (3.2%, 74.2%, and 97%, respectively). A cell line was derived from the patient at time of progression on entrectinib therapy. In vitro testing of the cell line demonstrated increased TKI responsiveness when treated with entrectinib and sotorasib. Signaling pathway analysis of the cell line showed entrectinib inhibited phosphorylation of ROS1 and AKT, but not ERK. Sotorasib alone or in combination with entrectinib resulted in decreased phosphorylation of ERK. In vitro testing suggested efficacy with the combination of entrectinib and sotorasib. However, in clinical follow up the patient’s disease demonstrated progression on the combination, likely related to KRAS G12C amplification. This case supports the need for broad molecular profiling for potential therapeutic and prognostic information to personalize therapy in patients with metastatic NSCLC. Citation Format: Katherine Priest, Anh Le, Dara Aisner, Amanuail Gebregzabheir, Hala Nijmeh, Melanie Mandell, Kurtis Davies, Emily O'Donnell, Robert C. Doebele, Liming Bao, Erin L. Schenk. Evolution of therapy resistance through acquired KRAS amplification in ROS1 fusion KRAS G12C double positive NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5233.

ROS1 TKIs entrectinib and crizotinib have significantly improved outcomes for ROS1+ lung adenocarcinoma patients. However, drug resistance inevitably develops, leading to disease progression. Approximately 1/3 of patients resistant to ROS1 TKIs demonstrate ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in most patients is unknown or poorly characterized. To model and characterize acquired resistance to ROS1 TKIs, we used patient-derived cell line CUTO28 (TPM3-ROS1) to generate an entrectinib-resistant derivative (CUTO28-ER) in vitro. We utilized several techniques to probe the mechanisms driving resistance: DNA and RNA sequencing (seq), fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. DNA seq of the ROS1 KD in CUTO28-ER failed to reveal mutations. CUTO28-ER cells displayed sensitivity to MET-selective TKIs in proliferation assays, and MAPK and AKT pathways were inhibited only with MET-selective TKI. RNA seq and western blot showed MET overexpression, and interphase FISH confirmed MET amplification compared to parental cells (MET:CEP7 ratio 4.2 vs 1.0). We substantiated in vitro findings in patient tissue, utilizing tumor samples at 2 different points of a single CD74-ROS1 NSCLC patient’s tumor progression. The first tumor sample did not display MET amplification while on ROS1 TKI (MET:CEP7 ratio 0.9, 3.4 copies of MET). However, the second tumor sample collected 5 months later displayed a MET:CEP7 ratio of 2.5 (9.5 copies of MET), indicating progression on ROS1 TKI was likely MET-driven. To determine prevalence of MET amplification in entrectinib-resistant ROS1+ NSCLC, we analyzed circulating tumor DNA (ctDNA) by FoundationOne Liquid CDx from patients with ROS1+ NSCLC in the STARTRK-2 entrectinib trial. Of 105 ROS1+ NSCLC patients with ctDNA analysis both at enrollment and progression, 2 (1.9%) displayed copy number amplification (CNA) of MET. Of these, 1 patient had no detectible CNA at study baseline but MET CNA gain by day 166 of entrectinib therapy and the other had detectable MET CNA gains both at baseline and at progression only 28 days later. Both received 3 lines of therapy prior to entrectinib, none of which targeted ROS1 or MET. In conclusion, we demonstrated MET gene amplification as a potential mechanism of resistance to ROS1 TKI entrectinib. The prevalence of MET amplification at resistance was 2 of 105, but may be greater than was detected in STARTRK-2 due to the sensitivity of ctDNA assays and challenges of measuring CNA in ctDNA. Utilization of the ROS1/MET TKI crizotinib or combination of entrectinib with capmatinib should be explored in patients with ROS1+ NSCLC that display MET CNA to overcome MET-driven resistance following entrectinib. Citation Format: Logan C. Tyler, Anh T. Le, Hala Nijmeh, Liming Bao, Timothy R. Wilson, Brian Simmons, David Chen, Robert C. Doebele. MET gene amplification is a mechanism of resistance to entrectinib in ROS1+ NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1103.

Clinical Practice Points • HER2 amplification is an acquired resistance mechanism in ALK-rearranged non–small cell lung cancer. • FISH analysis revealed a minor subclone of HER2-amplified cells before becoming the dominant resistance mechanism. • Combination of HER2 and ALK therapies may be an effective treatment approach for ALK-rearranged NSCLC with acquired HER2 amplification.

Additional file 1. List of complex karyotypes

Additional file 3. Comparison of clinical and molecular features of childhood CK-AML with ≤4 or ≥5 cytogenetic aberrations

Leber hereditary optic neuropathy (LHON) is a degenerative disease of the optic nerve associated with one of three mitochondrial DNA (mtDNA) m.3460G>A, m.11778G>A and m.14484T>C mutations. Although several procedures are available to genotype these mutations, quantitative approaches with rapid, low-cost and easy to handle advantages for three LHON mtDNA mutations are rarely reported. Here, we firstly developed a “one-step” tetra-primer amplification-refractory mutation system (T-ARMS) PCR for qualitative genotyping of three LHON mtDNA mutations. Subsequently, we established single, duplex and triplex TaqMan MGB probe-based fluorescence quantitative PCR (qPCR) assays to perform both qualitative and quantitative analyses of three LHON mtDNA mutations. Standard curves based on tenfold diluted plasmid standard exhibited high specificity and sensitivity, stable repeatability and reliable detectable ability of TaqMan probe qPCR assays without cross-reactivity upon probes combination. Moreover, by comparing with SYBR Green qPCR, we further validated the feasibility of the triplex-probe qPCR assay for the quantitative detection of mtDNA copy number in blood samples. In conclusion, our study describes a rapid, low-cost, easy to-handle, and high-throughput TaqMan-MGB probe qPCR assay to perform both qualitative and quantitative analysis of three primary LHON mtDNA mutations, offering a promising approach for genetic screening and testing of LHON mutations.

Although considered the same disease by 2016 WHO Classification, B-ALL and B-LBL show different clinicobiologic behavior, with B-ALL manifesting as disseminated disease and B-LBL as a localized mass. Distinction between the two is based on an arbitrary cutoff of 25% bone marrow involvement. We reviewed clinical, immunophenotypic, and cytogenetic data in B-lymphoblastic neoplasms of childhood to explain the differences. Performing a retrospective review of 126 cases of B-ALL and 18 cases of B-LBL in patients ≤18 years, revealed the following significant differences: younger age of presentation for leukemia; increased cytogenetic abnormalities in leukemia than lymphoma, specifically increased recurrent genetic abnormalities, with the exception of ploidy aberrancy; and the observation that unfavorable recurrent genetic abnormalities occurred in B-ALL and only favorable abnormalities in B-LBL. Down syndrome presented with leukemia only. Findings demonstrated that pediatric B-ALL and B-LBL exhibit dissimilar genomic profiles, suggesting possible differences in pathogenesis between the two closely-related neoplasms.

Conducting polymers tethered with molecular recognition elements are good candidates for biosensing applications such as detecting a target molecule with selectivity. We develop a new monomer, namely, 3,4-ethylenedioxythiophene bearing a pyridylboronic acid moiety (EDOT-PyBA), for label-free detection of sialic acid as a cancer biomarker. PyBA, which is known to show specific binding to sialic acid in acid conditions is used as a synthetic ligand instead of lectins. PyBA confirms the enhanced binding affinity for sialic acid at pH 5.0-6.0 compared with traditional phenylboronic acid. Poly(EDOT-PyBA) is electrodeposited on a planar glassy carbon electrode and the obtained film is successfully characterized by X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy, water contact angle measurements, and electrochemical impedance spectroscopy. The specific interaction of PyBA with sialic acid at the solution/electrode interface is detected by differential pulse voltammetry in a dynamic range 0.1-3.0 mM with a detection limit of 0.1 mM for a detection time of 3 min. The sensitivity covers the total level of free sialic acid in human serum and the assay time is the shorter than that of other methods. The poly(EDOT-PyBA) electrode successfully detects spiked sialic acid in human serum samples. Owing to its processability, mass productivity, and robustness, polythiophene conjugated with "boronolectin" is a candidate material for developing point-of-care and wearable biosensors.

Objectives Recent reports indicated that percutaneous coronary intervention (PCI) may be correlated with increased mortality in patients undergoing transcatheter aortic valve implantation (TAVI). Therefore, we performed a meta-analysis to determine the feasibility and safety of combined PCI in high-risk patients with severe aortic stenosis undergoing TAVI. Methods A comprehensive literature search was performed using PubMed, Embase and the Cochrane Central Register of Controlled trials through June 2016. Results Five clinical trials including 1634 patients were identified. The pooled analysis revealed no significant differences in 30-day all-cause mortality [odds ratio (OR) 1.25, 95% confidence interval (CI) 0.52-3.05; P = 0.62], 30-day cardiovascular mortality rate (OR 1.59, 95% CI 0.52-4.88; P = 0.41) and 1-year mortality rate (OR 1.16, 95% CI 0.85-1.59; P = 0.34) among the patients assigned to TAVI and those undergoing TAVI+PCI. The incidence of myocardial infarction (OR 2.96, 95% CI 1.03-8.45; P = 0.04) was slightly higher in the TAVI+PCI group. Other complications, such as stroke, kidney injury, bleeding and vascular complications, were not significantly increased in the TAVI+PCI group. Patients treated with a staged procedure of TAVI and PCI but not simultaneous TAVI+PCI showed higher 30-day all-cause mortality as compared to those undergoing isolated TAVI. Conclusions Combined TAVI+PCI showed similar rates of death from any cause at 30 days and 1 year as compared to isolated TAVI. Except for myocardial infarction, the rate of operative complications in the TAVI+PCI group was not detrimental as compared to the isolated TAVI group. The simultaneous treatment of significant coronary artery lesions may be preferred in selected patients undergoing TAVI.

Malva verticillata L. (M. verticillata) is an edible medicinal plant. Its dry, mature seeds are used in Traditional Mongolian Medicine. Nevertheless, detailed information regarding its chemical composition remains scarce. In this study, nineteen compounds were identified preliminarily using a UPLC-Q-TOF-MSE method. All of the compounds from M. verticillata are reported for the first time. M. verticillata is shown to be a rich source of phenolics, and the total phenolic content in the leaves, stems and seeds is 110.32 ± 3.45 mg GAE/g extract, 97.98 ± 4.19 mg GAE/g extract and 40.85 ± 7.63 mg GAE/g extract, respectively. The leaves are ideal for scavenging ABTS free radicals and possess ferric reducing antioxidant power, and the seeds are ideal for scavenging DPPH free radicals. M. verticillata is a rich source of natural antioxidants. These findings support the notion that M. verticillata is a functional food and can be used in nutraceutical applications.

Circulating cell-free tumor DNA (ctDNA) in the blood is DNA released from apoptotic, circulating, and living tumor cells. ctDNA is about 140 nt in length and has a half-life of about 1.5 h. ctDNA analysis provides a noninvasive means to assess the genetic profile of cancer in real time. With the advent of molecular technologies, including digital PCR and massively parallel sequencing (MPS), ctDNA analysis has shown promise as a highly sensitive and specific alternative to conventional tissue biopsy in cancer detection, longitudinal monitoring, and precision therapy. This review provides an overview of the latest development in our understanding of the biologic characteristics, detection methodologies, and potential clinical implications of ctDNA, as well as the challenges in translating ctDNA analysis from the research arena to patient care.

ROS1 tyrosine kinase inhibitors (TKIs) entrectinib and crizotinib have significantly improved outcomes for patients with ROS1 gene fusion positive (ROS1+) lung adenocarcinoma. However, drug resistance inevitably develops, leading to disease progression. Approximately one-third of patients resistant to ROS1 TKIs demonstrate on-target ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in the majority of patients remain unknown or poorly characterized. The purpose of this study was to model and characterize mechanisms of acquired ROS1 TKI resistance. To investigate resistance to ROS1 TKIs, we used patient-derived cell lines CUTO28 (TPM3-ROS1) and CUTO37 (CD74-ROS1) to generate drug-resistant derivatives to entrectinib (CUTO28-ER, CUTO37-ER) or crizotinib (CUTO28-CR,CUTO37-CR) in vitro. We utilized several techniques to probe the mechanisms driving resistance, including DNA and RNA sequencing, fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. DNA sequencing of the ROS1 KD in all resistant cell lines did not reveal any mutations. CUTO28-ER cells displayed sensitivity to MET-selective TKIs in cell proliferation assays. Consistent with MET dependency, MAPK and Akt pathways were inhibited by treatment with a MET-selective, but not a ROS1-selective, TKI. Western blot demonstrated MET overexpression and interphase FISH confirmed MET gene amplification (MET:CEP7 ratio 4.2) compared to parental cells (MET:CEP7 ratio 1.0). Notably, metaphase FISH revealed extrachromosomal DNA (ecDNA) amplification of MET. CUTO28-CR cells displayed sensitivity to Src family kinase (SFK) TKIs, and addition of pan-HER TKI afatinib resulted in synergistic inhibition of proliferation and downstream MAPK signaling. CUTO37-ER cells displayed sensitivity via proliferation and signaling to pan-HER inhibitor afatinib, and phosphorylation of EGFR Y845 consistent with Src-mediated activation of EGFR. Parental CUTO28 and CUTO37 cells were not sensitive to SFK or HER inhibition. Interphase FISH revealed modest EGFR gene amplification in CUTO28-CR cells—(EGFR:CEP7 ratio 2.1) compared to parental (EGFR:CEP7 ratio 1.0)—but not in CUTO37-ER or CUTO37-CR cells. HER2 gene amplification was not identified in any of the cell lines. Stimulation with epidermal growth factor (EGF) markedly enhanced MAPK signaling in CUTO28-CR, CUTO37-ER, and CUTO37-CR cells to a greater extent than in corresponding parental cells, consistent with EGFR or HER2 dependence. In conclusion, we demonstrated novel mechanisms of resistance to ROS1 TKIs entrectinib and crizotinib. These include ecDNA amplification of MET as an alternate oncogene driver and Src/EGFR axis as a driver of bypass signaling. Combination strategies with existing TKIs could be explored in patients with ROS1 TKI resistance. The discovery of ecDNA MET amplification is particularly intriguing; ecDNA is associated with more aggressive cancers and next generation sequencing analyses do not typically involve amplicon reconstruction to detect ecDNA. Citation Format: Logan C. Tyler, Anh T. Le, Hala Nijmeh, Liming Bao, Kristen Turner, Jason Christianson, Robert C. Doebele. Novel mechanisms of acquired TKI resistance in ROS1+ NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P076.

The prevalence and clinical relevance of KIT mutations in childhood core-binding factor (CBF) acute myeloid leukemia (AML) have not been well characterized. In this study, a total of 212 children with de novo AML were enrolled from a Chinese population and 50 (23.5%) of the patients were deemed CBF-AML. KIT mutations were identified in 30% of the CBF-AML cohort. The KIT mutations were clustered in exon 17 and exon 8, and KIT mutations in exons 8 and 17 were correlated with a shorter overall survival (OS) (5-year OS: 30.0 ± 14.5% vs. 73.0 ± 8.5%, p = .007) and event-free survival (EFS) (5-year EFS: 30.0 ± 14.5% vs. 73.0 ± 8.5%, p = .003). Multivariate analysis revealed KIT mutations as an independent risk factor in CBF-AML. Our results suggest that KIT mutations are a molecular marker for an inferior prognosis in pediatric CBF-AML.