A functional sgRNA-CRISPR screening method for generating murine RET and NTRK1 rearranged oncogenes.

CRISPR/Cas9 gene editing represents a powerful tool for investigating fusion oncogenes in cancer biology. Successful experiments require that sgRNAs correctly associate with their target sequence and initiate double stranded breaks which are subsequently repaired by endogenous DNA repair systems yielding fusion chromosomes. Simple tests to ensure sgRNAs are functional are not generally available and often require single cell cloning to identify successful CRISPR-editing events. Here, we describe a novel method relying on acquisition of IL3-independence in Ba/F3 cells to identify sgRNA pairs that generate oncogenic gene rearrangements of the Ret and Ntrk1 tyrosine kinases. The rearrangements were confirmed with PCR, RT-PCR and sequencing and Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNA pairs inducing the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) established from a Trim24-Ret positive tumor exhibited high in vitro sensitivity to the RET inhibitors LOXO-292 and BLU-667 and orthotopic TR.1 cell-derived tumors underwent marked shrinkage upon LOXO-292 treatment. Thus, the method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel, syngeneic murine oncogene-driven tumor models.

A Rapid, Functional sgRNA Screening Method for Generating Murine RET and NTRK1 Fusion Oncogenes.

CRISPR/Cas9 gene editing technology is an indispensable and powerful tool in the field of cancer biology. To conduct successful CRISPR-based experiments, it is crucial that sgRNAs generate their designed alterations. Here, we describe a simple and efficient sgRNA screening method for validating sgRNAs that generate oncogenic gene rearrangements. We used IL3-independence in Ba/F3 cells as an assay to identify sgRNA pairs that generate fusion oncogenes involving the Ret and Ntrk1 tyrosine kinases. We confirmed these rearrangements with PCR or RT-PCR as well as sequencing. Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNAs that catalyze the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) was established from a Trim24-Ret positive tumor that exhibited high in vitro sensitivity to RET-specific TKIs. Moreover, orthotopic transplantation of TR.1 cells into the left lung yielded well-defined tumors that shrank in response to LOXO-292 treatment. The method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel murine oncogene-driven tumor models.

Can electronic assessment tools improve the process of shared decision-making? A systematic review.

BACKGROUND: Patient involvement in decision-making plays a prominent role in improving the quality of healthcare. Despite this, shared decision-making is not routinely implemented. However, electronic assessment tools that capture patients' history, symptoms, opinions and values prior to their medical appointment are used by healthcare professionals during patient consultations to facilitate shared decision-making. OBJECTIVE: To assess the effectiveness of electronic assessment tools to improve the shared decision-making process. METHOD: A systematic review was conducted following PRISMA guidelines. Published literature was searched on MEDLINE, EMBASE and PsycINFO to identify potentially relevant studies. Data were extracted and analysed narratively. RESULTS: Seventeen articles, representing 4004 participants, were included in this review. The main findings were significant improvement in patient-provider communication and provider management of patient condition in the intervention group compared to the control group. In contrast, patient-provider satisfaction and time efficiency were assessed by relatively few included studies, and the effects of these outcomes were inconclusive. CONCLUSION: This review found that communication and healthcare professional's management of a patient's condition improves because of the use of electronic questionnaires. This is encouraging because the process of shared decision-making is reliant on high-quality communication between healthcare professionals and patients. IMPLICATIONS: We found that this intervention is especially important for people with chronic diseases, as they need to establish a long-term relationship with their healthcare provider and agree to a treatment plan that aligns with their values. More rigorous research with validated instruments is required.

A prospective observational study of the impact of an electronic questionnaire (ePAQ-PO) on the duration of nurse-led pre-operative assessment and patient satisfaction.

OBJECTIVE: Standard pre-operative assessment at our institution involves a comprehensive history and examination by a nurse practitioner. An electronic pre-operative assessment questionnaire, ePAQ-PO® (ePAQ, Sheffield, UK) has previously been developed and validated. This study aimed to determine the impact of ePAQ-PO on nurse consultation times and patient satisfaction in low-risk patients. METHODS: The duration of pre-operative assessment consultation was recorded for American Society of Anesthesiology physical classification 1 and 2 patients undergoing pre-operative assessment by an electronic questionnaire (ePAQ-PO group) and standard face-to-face assessment by a nurse practitioner (standard group). Patients were also asked to complete an eight-item satisfaction questionnaire. Eighty-six patients were included (43 in each group). RESULTS: After adjusting for the duration of physical examination, median (IQR [min-max]) consultation time was longer in the standard compared to the ePAQ-PO group (25 (18-33 [10-49]) min vs. 12 (8-17 [4-45]) min, respectively; p <0.001). Response rate for the satisfaction questionnaire was 93%. There was no significant difference in patient satisfaction scores (38/39 in standard group vs. 39/41 in ePAQ-PO group were fully satisfied with their pre-operative assessment; p = 0.494). CONCLUSION: Pre-operative assessment using ePAQ-PO is associated with a significant reduction of over 50% in the duration of the assessment without impacting on patient satisfaction.

Resistance to RET-Inhibition in RET-Rearranged NSCLC Is Mediated By Reactivation of RAS/MAPK Signaling.

Oncogenic rearrangements in RET are present in 1%-2% of lung adenocarcinoma patients. Ponatinib is a multi-kinase inhibitor with low-nanomolar potency against the RET kinase domain. Here, we demonstrate that ponatinib exhibits potent antiproliferative activity in RET fusion-positive LC-2/ad lung adenocarcinoma cells and inhibits phosphorylation of the RET fusion protein and signaling through ERK1/2 and AKT. Using distinct dose escalation strategies, two ponatinib-resistant LC-2/ad cell lines, PR1 and PR2, were derived. PR1 and PR2 cell lines retained expression, but not phosphorylation of the RET fusion and lacked evidence of a resistance mutation in the RET kinase domain. Both resistant lines retained activation of the MAPK pathway. Next-generation RNA sequencing revealed an oncogenic NRAS p.Q61K mutation in the PR1 cell. PR1 cell proliferation was preferentially sensitive to siRNA knockdown of NRAS compared with knockdown of RET, more sensitive to MEK inhibition than the parental line, and NRAS dependence was maintained in the absence of chronic RET inhibition. Expression of NRAS p.Q61K in RET fusion expressing TPC1 cells conferred resistance to ponatinib. PR2 cells exhibited increased expression of EGFR and AXL. EGFR inhibition decreased cell proliferation and phosphorylation of ERK1/2 and AKT in PR2 cells, but not LC-2/ad cells. Although AXL inhibition enhanced PR2 sensitivity to afatinib, it was unable to decrease cell proliferation by itself. Thus, EGFR and AXL cooperatively rescued signaling from RET inhibition in the PR2 cells. Collectively, these findings demonstrate that resistance to ponatinib in RET-rearranged lung adenocarcinoma is mediated by bypass signaling mechanisms that result in restored RAS/MAPK activation. Mol Cancer Ther; 16(8); 1623-33. ©2017 AACR.