The COVID-19 pandemic has had significant secondary impacts on healthcare services worldwide, including delays to non-essential procedures and surgeries. Early efforts to reduce the spread of COVID-19 across Canada resulted in up to 45% fewer visits to family physicians and medical specialists between March and June of 2020 (National Physician Database, 2020, Canadian Institute for Health Information). In the context of cancer care, this may have resulted in fewer new cancer diagnoses as a consequence of limited access to a family physician or diagnositic services, and delayed time to surgery or treatment.

PIK3CA (phosphatidylinositol-3 kinase) mutations have been found in multiple tumor sites including NSCLC among others, in the latter the frequency range between 2-7%. It has been related to cell growth, metabolism and survival as part of the PI3K-AKT-mTOR pathway. As part of this pathway, it may be amenable to targeted therapies which target mTOR.

We have previously reported increased survival in relapsed compared to de novo metastatic non-SCLC (NSCLC) patients. This study compared outcomes between real-world SCLC patients with extra-pulmonary metastasis at diagnosis (de novo) and at relapse in Alberta, Canada.

NSCLC is a disease of the elderly, with a median age at diagnosis of 70 years old. Yet elderly patients with NSCLC are under-represented in clinical trials, and studies of real-world treatment patterns have consistently identified lower rates of systemic therapy administration in this patient population. However there are clinical trial data to suggest that even the very elderly (age 80+) with advanced NSCLC can benefit from system therapy. We therefore looked at real-world patterns of treatment and survival in a cohort of patients aged 80 or older with metastatic NSCLC.

We have previously reported that rapid physical decline and poor performance status means that only a minority of patient with metastatic NSCLC receive systemic treatment. Over the last decade, treatment advances in the form of maintenance chemotherapy, targeted therapies and immunotherapy for advanced disease have shown benefit in randomized phase III trials and been adopted as standard of care. Nevertheless, there are conflicting reports of trends in overall survival. We therefore looked at real-world patterns of treatment and survival in a cohort of patients with metastatic NSCLC.

CCND2 (12p13) duplications and point mutation BRAF (7q34) p.V600E (c.1799T>A).MLPA analysis of control group showed normal results (fig2).Conclusion: The MLPA technique detected genomic abnormalities in seven MPE-LC samples, when at least 30% of tumor cells were present in the cytological examination.The findings of pathogenic variants in MPE-LC can be valuable to explore new targets for the treatment of lung cancer patients.

The driving principle behind precision medicine is to specifically target genetic variations that arise in tumorigenesis while leaving normal cells unaffected. Mutations in Ataxia Telangiectasia Mutated (ATM) may offer such a therapeutic target. ATM is mutated in approximately 12% of lung cancers and up to 40% of lung adenocarcinoma have been reported to lack ATM protein expression. ATM is an apex signaling kinase that responds to DNA-double strand breaks, playing a direct role in DNA repair as well as the initiation of signaling cascades that can lead to cell cycle arrest and apoptosis. We asked whether ATM-deficient human lung cancer cells are sensitive to the poly-ADP ribose polymerase (PARP) inhibitor olaparib, and investigated the mechanism of action of olaparib in these cells. We analyzed drug sensitivity for 61 lung adenocarcinoma cell lines from the Genomics of Drug Sensitivity in Cancer (GDSC) project and deleted ATM from lung adenocarcinoma A549 cells using CRISPR/Cas9. We determined the effects on cell viability using trypan blue exclusion and clonogenic survival assays. To investigate the mechanism of sensitivity of ATM-deficient cells to PARP and ATR inhibitors we used flow cytometry and cell viability assays as above. We observed a positive correlation between olaparib IC50 values and ATM mRNA expression. ATM mutant cell lines were more sensitive to olaparib compared to ATM wild-type cell lines or cell lines with amplified ATM. Additionally, ATM-deficient lung cancer cells were sensitive to olaparib, as are lung cancer cells (A549) with CRISPR/Cas9 deletion of ATM. Mechanistically, olaparib caused the temporary and reversible accumulation of G2 phase cells in ATM-deficient cells which manifested as a decrease in proliferation in both the trypan blue exclusion assay and clonogenic survival assay. Olaparib did not induce cell death in ATM-deficient cells, however cell death was induced when olaparib was used in combination with the ATR inhibitor VE-821. We show that olaparib acts as a cytostatic agent in ATM-deficient lung cancer cells, inducing a reversible and temporary growth arrest in G2 phase. Only when combined with the ATR inhibitor VE-821 was cell death observed and only in ATM-deficient cells. Our data suggest that patients with ATM-deficient lung cancer could benefit from combinatorial treatment with PARP and ATR inhibitors.

Background: Lung adenocarcinoma (LUAD) accounts for 40% of all lung cancer cases.Although driver mutations in the K-RAS oncogene occurs in 25% of all LUAD cases, to date, there are no available targeted therapies.Infiltrating neutrophils in LUAD are indicative of the worst survival outcomes.The C-X-C motif chemokine receptor 2 (CXCR2) mediates their recruitment to the tumour microenvironment where they promote a pro-tumorigenic environment.CXCR2 ligand expression is higher in KRAS-driven LUAD compared to the other most frequently mutated oncogenes.Therefore, we hypothesize that K-RASdriven LUAD may be the best candidate for a CXCR2 targeted treatment strategy.Method: The PREdiction of Clinical Outcomes from Genomic Profiles (PRECOG) is a dataset of gene expression and survival outcome.The dataset includes data from approximately 18 000 human patients with 39 different malignancies.The dataset was used to determine whether high neutrophil infiltration, CXCR2 expression and CXCR2 ligand expression were associated with poor survival outcomes in LUAD.A 100 patient LUAD tissue microarray was built and stained for neutrophil elastase and CXCR2 by immunohistochemistry. Kaplan-Meier curves were used determine the effect of high neutrophil or CXCR2+ cell infiltration in the LUAD tumour microenvironment on survival outcome.The Cancer Cell Line Encyclopedia (CCLE) is an online dataset that provides gene expression and genotype data from 947 human cancer cell lines (36 cancer types).Expression data of all LUAD cell lines (n¼70) from CCLE was obtained for all known CXCR2 ligands.The expression of CXCR2 ligands in K-RAS, EGFR, ALK and ROS-1-driven LUAD cell lines was compared.Microfluidics devices were used to compare the neutrophil recruitment to K-RAS, EGFR, ALK and ROS-1-driven LUAD cell lines.The neutrophil recruitment to each of the cell lines was compared in the presence and absence of CXCR2 inhibition.Result: Using the PRECOG dataset, we found that CXCR2 expression in neutrophils is at least 18-fold greater than its expression in other immune cell types.Using all the LUAD cell lines (n¼70) available on the CCLE, we found that K-RAS-driven LUAD is the highest CXCR2 ligand expresser as compared to EGFR, ALK and ROS1-driven LUAD.Moreover, using PRECOG, we found that poorer survival outcome is associated with high expression of eight out of nine known CXCR2 ligands (p <0.05).In addition, high neutrophil infiltration in LUAD is associated with the worst survival outcome compared to other immune cell infiltrates (p <0.001).In accordance with the PRECOG data, the presence of infiltrating neutrophils in a 100 patient LUAD tissue microarray is associated with poorer survival outcome when compared to patients with no infiltrating neutrophils (p <0.05).Neutrophil migration to K-RAS, EGFR, ALK and ROS1-driven LUAD cell lines was examined in microfluidics devices and found to be highest in K-RAS-driven LUAD.CXCR2 inhibition reduced neutrophil migration only in K-RAS-driven lung adenocarcinoma (p <0.05).Conclusion: CXCR2 inhibition could be an exciting potential targeted treatment for patients with K-RASdriven LUAD.CXCR2 inhibition is in clinical trials for metastatic melanoma, pancreatic, breast and head and neck cancer.Current evidence suggests that CXCR2 inhibition is safe and tolerable.

Immunotherapies targeted against PD-L1 and PD-1 have caused a paradigm shift in the treatment of NSCLC, and PD-L1 protein expression has emerged as a standard diagnostic biomarker that predicts which patients are more likely to respond to immunotherapy. However, the use of PD-L1 protein expression as a biomarker is complicated by differences in PD-L1 antibodies, immunohistochemistry methods and platforms, pathologist scoring, and positivity cut-points. We propose that using RNA-sequencing (RNA-seq) methodologies will be an equally reliable approach to determine PD-L1 expression within a tumour and will yield a greater depth of biomarker information than PD-L1 IHC alone. We performed quantitative immunohistochemistry (qIHC) on 262 resected stage I-III formalin-fixed paraffin-embedded (FFPE) NSCLC patient samples registered in the Glans-Look Lung Cancer Research (GLR) database using the commercially available E1L3N PD-L1 antibody (Cell Signalling Technologies). Staining intensity was quantified and used to establish a positivity threshold that was subsequently used to define positivity cut-points of <1%, 1%-49%, and >50% (as used for determining treatment eligibility for Pembrolizumab). We performed single-end RNA-sequencing on FFPE samples from the same GLR patient cohort. Raw counts were normalized to counts-per-million for use in our analyses. We compared the PD-L1 mRNA expression to the PD-L1 protein staining intensity across the tissue core and found a significant correlation (p<0.001, Spearman's rho=0.538). We also found significant correlation between PD-L1 mRNA expression and the percent-positivity score determined by qIHC (p<0.001, Spearman's rho=0.605), which was particularly apparent when comparing PD-L1 mRNA expression between cut-point groups where expression was significantly higher in the 1%-49% and >50% groups. Interestingly, we also found moderate, yet significant correlation between PD-1 mRNA expression and both PD-L1 protein staining intensity and percent positivity (p<0.001, Spearman's rho=0.437 and 0.415 respectively), and we were able to identify several differentially expressed genes between the PD-L1 positive and negative groups. Given the high degree of correlation between PD-L1 mRNA expression and PD-L1 protein staining and positivity, RNA-seq can be a viable option for assessing candidacy for immunotherapy. In addition to the wealth of supplementary data on important biomarkers, RNA-seq offers the possibility for using non-invasive procedures such as liquid biopsy to measure PD-L1 levels in a sequential, objective fashion.

Histopathological distinction between non-small cell lung cancer subtypes is still relevant in the age of targeted therapy due to the relatively low number of patients with actionable molecular alterations such as EGFR mutations or ALK rearrangements in squamous cell carcinomas. In addition, for patients without these alterations, the choice of most effective chemotherapy regimens is often based upon non-squamous vs squamous cell histology. However, histopathology scoring typically requires resected or biopsied tissue to be fixed, sliced, stained, and subjectively scored by a pathologist. Here we endeavor to demonstrate that subtle variances in gene expression within NSCLC patient samples can be used to classify the cancer subtype with a high degree of accuracy to pathology classification. RNA extracted from 46 resected NSCLC cases (34 stage IIA/B, 12 stage IIIA/B; primarily adenocarcinomas and squamous cell carcinomas) were profiled using a transcriptome microarray platform (Illumina) and analyzed and compared to normal tissue using several Bioconductor R package pathway analysis tools and DAVID Bioinformatics Resources 6.8. Expression profiles were grouped by principle component score in a blinded manner. Clustering samples based on the top 10% most variable genes resulted in a highly accurate separation of adenocarcinoma samples and squamous cell carcinoma samples, with only one case of squamous cell carcinoma being misclassified as adenocarcinoma. The altered pathways that differentiated these samples included p53 signalling and PI3K-Akt signaling pathways, Cell adhesion molecules (CAM), ECM-receptor interactions, Wnt signalling and several other key pathways. Due to the successes of targeted therapeutic approaches, evaluating patient biopsies or other samples for EFGR mutations and ALK rearrangements are currently the standard of care for advanced-stage NSCLC. Immunohistochemical staining for the purposes of subtyping NSCLC can aid in developing treatment strategies for patients without these molecular alterations, but this can significantly reduce the quantity of tissue available for subsequent molecular tests. Based on our data, we believe that expression variances in a relatively small pool of genes and pathway analysis is sufficient to accurately predict the subtype of NSCLC, or at least narrow the number of cases requiring input from a pathologist to a small number of more difficult cases.

Ataxia telangiectasia-mutated (ATM) is a critical first responder to DNA damage in the cell, but despite being one of the most mutated genes in lung cancer, no specific mutation hotspots have been linked with disease development. Our own quantitative analysis of ATM protein levels in patient samples suggests that ATM is lost in 20-25% of cases and that this loss correlates with poor overall survival and increased response to adjuvant chemotherapy treatments. We believe that this may be the result of increased genomic instability within the cancer cells caused by a lack of adequate DNA repair. Given that ATM-deficient cancers may have higher genetic instability, and that ATM is so highly mutated in lung cancer, we sought to quantify the relationship between ATM mutations and genomic instability, as measured by somatic mutation burden.

Platinum based antineoplastic therapies (platins) are a first line treatment for non-small cell lung cancer (NSCLC) that generate DNA breaks and stimulate DNA damage response pathways. An inability to repair damage generated by these agents leads to cytotoxicity and cell death. A key mediator of the DNA damage response is ataxia telangiectasia mutated (ATM), an activator of downstream targets involved in DNA repair, cell cycle arrest, and apoptosis. Our lab has demonstrated that cell lines lacking ATM show increased sensitivity to platins. We hypothesize that platin exposure will activate ATM and that cells deficient in ATM will be innately sensitive to platins. Here we assess the molecular action of ATM in response to platins to determine if ATM-deficiency is predictive of platin sensitivity. ATM status was determined in five NSCLC cell lines using western blotting and RT-qPCR. Cell lines were treated with varying concentrations cisplatin, carboplatin and oxaliplatin for 18-hours and assessed for ATM phosphorylation by western blot. Additionally, downstream targets of ATM (KAP-1, p53, and gamma-H2AX) were investigated to determine ATM pathway activation. Knockdown cell lines were generated using shRNA to ATM before testing for IR and cisplatin sensitivity using clonogenic assay. ATR and ATM inhibitors were tested on knockdown cell lines to investigate pathway response differences after cisplatin and IR. NSCLC cell lines NCI-H226, NCI-H460, and NCI-H522 were found to be ATM-proficient whereas cell lines NCI-H23 and NCI-H1373 were found to be ATM-deficient. ATM-proficient cell lines demonstrated an increased level of phosphorylated-ATM in response to treatments with cisplatin, carboplatin, and oxaliplatin. ATM knockdown cell lines were found to have increased sensitivity to IR, however analysis of cisplatin sensitivity was inconclusive with only 1 out of 4 showing increased sensitivity. ATR inhibition in combination with cisplatin caused a large increase in DNA damage response from ATM and DNA-PKcs suggesting an avenue for synthetic lethality. It is clear that platin exposure induced an ATM mediated signaling response and that cells lacking ATM showed deficiencies in the phosphorylation of key downstream targets. Cells deficient in ATM may therefore be more susceptible to platin therapy due to an impaired DNA repair response. However, the predictive capabilities of ATM loss for platin sensitivity is still unclear. This data suggests that individuals with low or non-functioning ATM may be candidates for precision low dose therapies that exploit this deficiency.

Introduction: We have shown that mantle cell lymphoma and gastric cancer cell lines deficient in the DNA repair protein ATM display increased sensitivity to ionizing radiation and a greater susceptibility to PARP inhibitor-induced synthetic lethality–a phenotype we have labeled “ATMic”. Since this has potential clinical application, it will be important to be able to reliably identify ATMic tumors so that appropriate precision therapy can be offered to patients. We compared the utility of various assays to identify ATMicity in a series of Non-small cell lung cancer (NSCLC) cell lines.