Supplementary Figures 1-2, Table 1 from FMS-Like Tyrosine Kinase 3–Internal Tandem Duplication Tyrosine Kinase Inhibitors Display a Nonoverlapping Profile of Resistance Mutations In vitro

Remodeling of the bone marrow microenvironment in chronic inflammation and in aging reduces hematopoietic stem cell (HSC) function. To assess the mechanisms of this functional decline of HSC and find strategies to counteract it, we established a model in which the Sfrp1 gene was deleted in Osterix+ osteolineage cells (OS1Δ/Δ mice). HSC from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the reactive oxygen species pathway and reduced single-cell proliferation. Interestingly, not only was the protein level of Catenin beta-1 (bcatenin) elevated, but so was its association with the phosphorylated co-activator p300 in the nucleus. Since these two proteins play a key role in promotion of differentiation and senescence, we inhibited in vivo phosphorylation of p300 through PP2A-PR72/130 by administration of IQ-1 in OS1Δ/Δ mice. This treatment not only reduced the b-catenin/phosphop300 association, but also decreased nuclear p300. More importantly, in vivo IQ-1 treatment fully restored HSC repopulating activity of the OS1Δ/Δ mice. Our findings show that the osteoprogenitor Sfrp1 is essential for maintaining HSC function. Furthermore, pharmacological downregulation of the nuclear b-catenin/phospho-p300 association is a new strategy to restore poor HSC function.

e13594 Background: Facilitating personalized medicine and applying precision oncology in treating breast cancer (BC) requires in vitro model systems that mimic the disease's heterogeneity and the clinical response to cancer treatment. The capability of patient-derived organoids (PDOs) to serve as a paramount model in this regard has been shown. Nonetheless, offering this model in a clinically relevant timeframe to support clinical decisions persists challenging. Methods: To examine the feasibility of PDOs as a precision oncology platform to guide treatment decisions in the clinic, we are conducting a prospective exploratory study to establish PDOs from core needle biopsies and surgical samples and evaluate Onco-PDO drug testing in a short timeframe (10-14 days) after biopsy. In addition, PDOs are further expanded and characterized histologically, genomically, and biobanked. More importantly, the clinical response of patients is monitored and correlated with the PDO drug response. Results: To date, 85 tumor samples from the primary tumor (n=64) and lymph node metastases (n=21) were received from 73 patients recruited in our ongoing prospective exploratory study in the Invitrocue Munich laboratory. The median age stands at 51 years (range 28-85). The success rate of PDO generation holds at 90%. Up to now, the Onco-PDO test has been successfully performed in 75% of cases (n=64) and failed in 25% (n=21). A broad spectrum of standard-of-care chemotherapeutic and targeted drugs in BC were tested, resulting in 942 readouts. The heatmap of drug screening indicates the highest sensitivity to anthracyclines such as epirubicin (E) and doxorubicin (D) as a single agent or in combination with cyclophosphamide (EC and DC). In other drugs a higher variability in the range of drug sensitivity has been observed: i.e., paclitaxel (T; 0-98%), carboplatin (C; 0-99%), and the combination (TC; 0-99%), as well as 5-FU (0-100%) and vinorelbine (0-95%). Thus far, the analysis of clinical correlation could be performed in patients who received epirubicin resulting in a significant difference (p=0.03) in the IC-50 of epirubicin between responder and non-responder patients. Conclusions: The prospective exploratory study using Onco-PDO test by Invitrocue could overcome the previous challenges concerning the implementation of PDOs in clinical practice by standardizing the methodology for PDO generation especially from small starting material such as core needle biopsies and drug testing more rapidly and efficiently, leading to providing results within a clinically relevant timeframe of 14 days after obtaining a biopsy. The onco-PDO test might serve as an ideal test enabling precision oncology by identifying effective treatments and sparing unnecessary side effects from ineffective treatments.

BACKGROUND:Mutations in cKIT or PDGFRA are found in up to 90% of patients with gastrointestinal stromal tumors (GISTs). Previously, we described the design, validation, and clinical performance of a digital droplet (dd)PCR assay panel for the detection of imatinib-sensitive cKIT and PDFGRA mutations in circulating tumor (ct)DNA. In this study, we developed and validated a set of ddPCR assays for the detection of cKIT mutations mediating resistance to cKIT kinase inhibitors in ctDNA. In addition, we cross-validated these assays using next generation sequencing (NGS).METHODS:We designed and validated five new ddPCR assays to cover the most frequent cKIT mutations mediating imatinib resistance in GISTs. For the most abundant imatinib-resistance-mediating mutations in exon 17, a drop-off, probe-based assay was designed. Dilution series (of decreasing mutant (MUT) allele frequency spiked into wildtype DNA) were conducted to determine the limit of detection (LoD). Empty controls, single wildtype controls, and samples from healthy individuals were tested to assess specificity and limit of blank (LoB). For clinical validation, we measured cKIT mutations in three patients and validated results using NGS.RESULTS:Technical validation demonstrated good analytical sensitivity, with a LoD ranging between 0.006% and 0.16% and a LoB ranging from 2.5 to 6.7 MUT fragments/mL. When the ddPCR assays were applied to three patients, the abundance of ctDNA in serial plasma samples reflected the individual disease course, detected disease activity, and indicated resistance mutations before imaging indicated progression. Digital droplet PCR showed good correlation to NGS for individual mutations, with a higher sensitivity of detection.CONCLUSIONS:This set of ddPCR assays, together with our previous set of cKIT and PDGFRA mutations assays, allows for dynamic monitoring of cKIT and PDGFRA mutations during treatment. Together with NGS, the GIST ddPCR panel will complement imaging of GISTs for early response evaluation and early detection of relapse, and thus it might facilitate personalized decision-making.

Supplementary Data from Histone Deacetylase Inhibitors Induce a Very Broad, Pleiotropic Anticancer Drug Resistance Phenotype in Acute Myeloid Leukemia Cells by Modulation of Multiple ABC Transporter Genes

Supplementary Tables 1-2, Figures Legends 1-4 from Stromal Niche Cells Protect Early Leukemic FLT3-ITD+ Progenitor Cells against First-Generation FLT3 Tyrosine Kinase Inhibitors

Investigation of dynamics of tumor rejection mediated by TCR2.5D6-transduced TCM to determine biologically critical imaging time points in an ML2-based myeloid sarcoma model

Determination of different tumor rejection kinetics according to tumor size

Supplementary Figures 1-4 from Stromal Niche Cells Protect Early Leukemic FLT3-ITD+ Progenitor Cells against First-Generation FLT3 Tyrosine Kinase Inhibitors

PDF file - 96K, FLT uptake is a measure for nonresponse to HSP90 inhibition in NPM-ALK+ overexpressing lymphoma cells in vitro

PDF File - 120K, BCR-ABL kinase domain mutations recovered in the sequential screen with IC50-values for imatinib, dasatinib and nilotinib.

Supplementary Material and Figure Legends

Supplementary Figure 1, Tables 1-7 from ADAM17 Regulates Epidermal Growth Factor Receptor Expression through the Activation of Notch1 in Non–Small Cell Lung Cancer

PDF file - 51K, Effect of everolimus on SUDHL-1 cells in vitro

Supplementary Data from Functional Analysis of Epidermal Growth Factor Receptor (EGFR) Mutations and Potential Implications for EGFR Targeted Therapy

Supplementary Data - PDF file 158K, Supplementary Table S1. Target mutations and ligation probes for mutation specific and wild type CKIT or PDGFRA hybridisation. Patient numbers corresponding to Figure 2, their specific tumor/target mutations and the sequences of the hybridisation probes. Supplementary Table S2. Overview of test results in relation to clinical setting for patients with active disease. Allocation of test results corresponding to Table 2 and to patient numbers corresponding to Figure 2. Supplementary Figure S1. LINE1 levels of GIST patients in complete remission versus progression and relapse. Shown are the median LINE1 fragment numbers in 2 microl plasma DNA

Investigation of uptake by biodistribution and autoradiography as well as stability of 89Zr-aTCRmu-F(ab')2 within a clinically relevant ML2-based myeloid sarcoma model of adoptive T-cell transfer

PDF file - 96K, FLT uptake is a sensitive measure for response to HSP90 inhibition in NPM-ALK overexpressing lymphoma cells in vitro